CX3CR1-binding polypeptides comprising immunoglobulin single variable domains

ABSTRACT

The present invention relates to CX3CR1-binding polypeptides, in particular polypeptides comprising specific immunoglobulin domains. The invention also relates to nucleic acids encoding such polypeptides; to methods for preparing such polypeptides; to host cells expressing or capable of expressing such polypeptides; to compositions comprising such polypeptides; and to uses of such polypeptides or such compositions, in particular for prophylactic, therapeutic and diagnostic purposes.

FIELD OF THE INVENTION

The present invention relates to CX3CR1-binding polypeptides, inparticular polypeptides comprising specific immunoglobulin domains. Theinvention also relates to nucleic acids encoding such polypeptides; tomethods for preparing such polypeptides; to host cells expressing orcapable of expressing such polypeptides; to compositions comprising suchpolypeptides; and to uses of such polypeptides or such compositions, inparticular for prophylactic, therapeutic and diagnostic purposes.

BACKGROUND OF THE INVENTION

CX3CR1 is a G-protein coupled integral membrane protein, which is achemokine receptor. It is predominantly expressed on cell types such asmonocytes, dendritic cells and T cells that have been associated withthe initiation and progression of atherosclerotic plaques. It isupregulated on monocytes by oxidized lipids and mediates migration ofthese cells into and survival within plaques. Its unique ligandfractalkine (FKN) is expressed on the surface of vascular endothelialand smooth muscle cells in lesions where it modulates leukocyteadhesion. Fractalkine is also released into the circulation byproteolytic cleavage where it functions as a chemotactic agent.

In humans, a CX3CR1 variant (V2491/T280M) with decreased activity hasbeen shown to be associated with a lower risk of cardiovascular disease(coronary heart disease, cerebrovascular disease or peripheral vasculardisease)(McDermott, 2001; Circ Res 89:401), coronary artery disease(angiographic evidence of stenosis) (McDermott, 2003; J. Clin. Invest.111:1241), and carotid artery occlusive disease (Ghilardi, 2004; Stroke35:1276). CX3CR1 co-localized with fractalkine which showed enhancedimmunostaining by polyclonal antibodies within atherosclerotic plaques(Wong, 2002 Cardiovasc. Path. 11:332). No fractalkine staining wasobserved in non-plaque arterial regions.

Several independent mouse genetic studies have shown a beneficial effectof CX3CR1 deficiency on atherosclerosis. A reduction in lesion area inthe aortic arch and thoracic aorta as well as a decrease inmonocyte/macrophage accumulation in plaques was seen in twoindependently derived strains of CX3CR1^(−/−) apoE^(−/−) mice fed a highfat diet (Combadière, 2003; Circulation, 107:1009, Lesnik, 2003; J.Clin. Invest. 111:333).

This shows that CX3CR1 is involved in cardiovascular diseases and themodulation of its activity could provide promising therapies. There istherefore a need for antagonist molecules against CX3CR1 with beneficialpharmacological properties, which can be used as therapeutic agents totreat diseases, in particular cardiovascular diseases in humans.

Accordingly, one aim of the present invention is to provide anti-CX3CR1antagonist molecules, in particular anti-CX3CR1 antagonist molecules,which have high binding affinity to CX3CR1.

A further aim of the present invention is to provide anti-CX3CR1antagonist molecules, which have high specificity for CX3CR1.

A further aim of the present invention is to provide anti-CX3CR1antagonists, which have potent activity.

A further aim of the present invention is to provide anti-CX3CR1antagonists, which have a favorable bioavailability and half-life.

A further aim of the present invention is to provide anti-CX3CR1antagonists, which have favorable biophysical properties.

Further aims of the present invention include combinations of any of theaims set forth above.

SUMMARY OF THE INVENTION

The invention provides polypeptides which bind to human CX3CR1 and arecapable of blocking the binding of human fractalkine to human CX3CR1. Inone aspect, the polypeptide is an immunoglobulin comprising anantigen-binding domain comprising three complementarity determiningregions CDR1, CDR2 and CDR3, wherein said immunoglobulin binds to humanCX3CR1 and is capable of blocking the binding of human fractalkine tohuman CX3CR1. In a further aspect, the polypeptide comprises one or moreanti-CX3CR1 immunoglobulin single variable domain, wherein saidpolypeptide is capable of blocking the binding of human fractalkine tohuman CX3CR1.

In one aspect, a polypeptide of the present invention is characterizedby one or more of the following properties:

-   -   Bind with high affinity to human CX3CR1;    -   Block the binding of soluble fractalkine to human CX3CR1;    -   Inhibit fractalkine induced chemotaxis;    -   Inhibit fractalkine induced human CX3CR1 receptor        internalization;    -   Cross-react with cyno CX3CR1 within 10-fold of E/IC₅₀ for human        CX3CR1 for binding and functional inhibition.

In a further aspect, a polypeptide of the present invention comprises ananti-CX3CR1 immunoglobulin single variable domain and further comprisesa half-life extending moiety, for example an albumin binding moiety, apolyethylene glycol molecule or a Fc domain. In a further aspect, apolypeptide of the present invention comprises two or more anti-CX3CR1immunoglobulin single variable domains. In one aspect, the twoanti-CX3CR1 immunoglobulin single variable domains are covalently linkedby a linker peptide. In one aspect, the two anti-CX3CR1 immunoglobulinsingle variable domains in a polypeptide of the present invention havethe same amino acid sequence. In another aspect, the two anti-CX3CR1immunoglobulin single variable domains in a polypeptide of the presentinvention have different amino acid sequences. In one aspect, apolypeptide of the present invention comprises two anti-CX3CR1immunoglobulin single variable domains and further comprises a half-lifeextending moiety, for example an albumin binding moiety, a polyethyleneglycol molecule or a Fc domain. In one aspect, a polypeptide of thepresent invention comprises a first anti-CX3CR1 immunoglobulin singlevariable domain covalently linked to an albumin binding moiety by afirst linker peptide, wherein said albumin binding moiety is furthercovalently linked to a second anti-CX3CR1 immunoglobulin single variabledomain by a second linker peptide.

In one aspect, a polypeptide of the present invention comprises ananti-CX3CR1 immunoglobulin single variable domain covalently linked to aFc domain by a linker peptide. In one aspect, such polypeptidecomprising an anti-CX3CR1 immunoglobulin single variable domaincovalently linked to a Fc domain by a linker peptide is provided as adimer, for example through disulfide bridges. The polypeptides of thepresent invention are used for the prevention, treatment, alleviationand/or diagnosis of CX3CR1-associated diseases, disorders or conditions,in particular cardiovascular diseases, such as atherosclerosis.

In a further aspect, the present invention provides:

Embodiment 1: An immunoglobulin comprising an antigen-binding domaincomprising three complementarity determining regions CDR1, CDR2 andCDR3, wherein said immunoglobulin binds to human CX3CR1 and is capableof blocking the binding of human fractalkine to human CX3CR1.

Embodiment 2: A polypeptide comprising one or more anti-CX3CR1immunoglobulin single variable domain, wherein said polypeptide iscapable of blocking the binding of human fractalkine to human CX3CR1.

Embodiment 3: A polypeptide according to embodiment 2, wherein saidanti-CX3CR1 immunoglobulin single variable domain consists essentiallyof four framework regions (FR1, FR2, FR3 and FR4) and threecomplementary determining regions (CDR1, CDR2 and CDR3).

Embodiment 4: A polypeptide according to embodiment 3, wherein saidanti-CX3CR1 immunoglobulin single variable domain has the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Embodiment 5: A polypeptide according to any one of embodiments 2 to 4,wherein said anti-CX3CR1 immunoglobulin single variable domain is anantibody domain.

Embodiment 6: A polypeptide according to embodiment 5, wherein saidanti-CX3CR1 immunoglobulin single variable domain is a VH, VL, VHH,camelized VH, or VHH that is optimized for stability, potency,manufacturability and/or similarity to human framework regions.

Embodiment 7: A polypeptide according to any one of embodiments 1 to 6,wherein said polypeptide has an affinity to human CX3CR1 at:

-   -   a) an EC50 of less than or equal to 10 nM, less than or equal to        5 nM, less than or equal to 2.5 nM or less than or equal to 1        nM, as determined by cell binding FACS; or    -   b) an IC50 of less than or equal to 10 nM, less than or equal to        5 nM, less than or equal to 2.5 nM or less than or equal to 1        nM, as determined by competition FACS.

Embodiment 8: A polypeptide according to any one of embodiments 1 to 7,wherein said polypeptide blocks the binding of human fractalkine tohuman CX3CR1 at an IC50 of less than or equal to 300 nM, or less than orequal to 100 nM, or less than or equal to 20 nM, or less than or equalto 10 nM, or less than or equal to 5 nM, or less than or equal to 2.5 nMor less than or equal to 1 nM.

Embodiment 9: A polypeptide according to any one of embodiments 1 to 8,wherein said polypeptide inhibits fractalkine induced chemotaxismediated by human CX3CR1 at an IC₅₀ of less than or equal to 500 nM, orof less than or equal to 100 nM, or of less than or equal to 75 nM, orof less than or equal to 50 nM, or less than or equal to 10 nM or lessthan or equal to 5 nM.

Embodiment 10: A polypeptide according to any one of embodiments 1 to 9,wherein said polypeptide inhibits fractalkine internalization mediatedby human CX3CR1 at an IC₅₀ of less than or equal to 10 nM, or less thanor equal to 5 nM or or less than or equal to 1 nM.

Embodiment 11: A polypeptide according to any one of embodiments 3 to10, wherein said CDR3 has the amino acid sequence ofAsp-Xaa1-Arg-Arg-Gly-Trp-Xaa2-Xaa3-Xaa4-XaaS (SEQ ID NO: 197), wherein:

-   -   Xaa1 is Pro, Ala or Gly;    -   Xaa2 is Asp or Asn;    -   Xaa3 is Thr or Ser;    -   Xaa4 is Arg, Lys, Ala or Gly; and    -   Xaa5 is Tyr or Phe.

Embodiment 12: A polypeptide according to any one of embodiments 3 to11, wherein:

a)

-   -   Xaa1 is Pro, Ala or Gly;    -   Xaa2 is Asp or Asn;    -   Xaa3 is Thr;    -   Xaa4 is Arg or Lys; and    -   Xaa5 is Tyr,        and/or        b) wherein said CDR3 is selected from any of SEQ ID No's:        186-190.

Embodiment 13: A polypeptide according to any one of embodiments 3 to12, wherein said CDR3 has the amino acid sequence ofAsp-Pro-Arg-Arg-Gly-Trp-Asp-Thr-Arg-Tyr (SEQ ID NO: 186).

Embodiment 14: A polypeptide according to any one of embodiments 3 to10, wherein:

-   -   i) said CDR1:        -   a) has the amino acid sequence of SEQ ID NO: 141;        -   b) has an amino acid sequence that has at least 80% amino            acid identity with the amino acid sequence of SEQ ID NO:            141;        -   c) has an amino acid sequence that has 2, or 1 amino acid(s)            difference with the amino acid sequence of SEQ ID NO: 141,            wherein            -   at position 2 the S has been changed into T, or G;            -   at position 6 the S has been changed into R;            -   at position 7 the N has been changed into T; and/or            -   at position 9 the M has been changed into K; or        -   d) has an amino acid sequence selected from any one of SEQ            ID NO's: 141-145 and 213;    -   ii) said CDR2:        -   a) has the amino acid sequence of SEQ ID NO: 164;        -   b) has an amino acid sequence that has at least 70% amino            acid identity with the amino acid sequence of SEQ ID NO:            164;        -   c) has an amino acid sequence that has 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 164, wherein            -   at position 1 the G has been changed into A, L, V or S;            -   at position 3 the N has been changed into D, S, Q, G or                T;            -   at position 4 the S has been changed into T, K, G or P;            -   at position 5 the V has been changed into A;            -   at position 6 the G has been changed into D;            -   at position 7 the I has been changed into T, or V;            -   at position 8 the T has been changed into A; and/or            -   at position 9 the K has been changed into R; or        -   d) has an amino acid sequence selected from any one of SEQ            ID NO's: 162-175 and 214-221; and    -   iii) said CDR3:        -   a) has the amino acid sequence of SEQ ID NO: 186;        -   b) has an amino acid sequence that has at least 70% amino            acid identity the amino acid sequence of SEQ ID NO: 186;        -   c) has an amino acid sequence that has 3, 2, or 1 amino            acid(s) difference with the amino acid sequences of SEQ ID            NO: 186, wherein            -   at position 2 the P has been changed into A, or G;            -   at position 7 the D has been changed into N; and/or            -   at position 9 the R has been changed into K; or        -   d) has an amino acid sequence selected from any one of SEQ            ID NO's: 186-190.

Embodiment 15: A polypeptide according to any one of embodiments 3 to10, wherein

-   -   i) said CDR1 has the amino acid sequence of SEQ ID NO: 146;    -   ii) said CDR2 has an amino acid sequence that a) has at least        90% amino acid identity with the amino acid sequence of SEQ ID        NO: 176 or b) has the amino acid sequence of SEQ ID NO: 176 or        177; and    -   iii) said CDR3 has the amino acid sequence of SEQ ID NO: 191.

Embodiment 16: A polypeptide according to any one of embodiments 3 to10, wherein

-   -   i) said CDR1:        -   a) has the amino acid sequence of SEQ ID NO: 147; or        -   b) has an amino acid sequence that has 6, 5, 4, 3, 2, or 1            amino acid(s) difference with the amino acid sequence of SEQ            ID NO: 147, wherein            -   at position 1 the G has been changed into K, R, or A;            -   at position 2 the T has been changed into I, P, S or L;            -   at position 3 the I has been changed into V, or T;            -   at position 4 the F has been changed into L;            -   at position 5 the S has been changed into R, or D;            -   at position 6 the N has been changed into S, T, or D;                and/or            -   at position 7 the N has been changed into T, or Y; or        -   c) has an amino acid sequence selected from any one of SEQ            ID NO's: 147-161;    -   ii) said CDR2:        -   a) has the amino acid sequence of SEQ ID NO: 179; or        -   b) has an amino acid sequences that has 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 179, wherein            -   at position 3 the S has been changed into T, or G;            -   at position 4 the N has been changed into S, or I;            -   at position 5 the S has been changed into T;            -   at position 6 the G has been changed into Y; and/or            -   at position 8 the T has been changed into A; or        -   c) has an amino acid sequence selected from any one of SEQ            ID NO's: 178-185; and    -   iii) said CDR3:        -   a) has the amino acid sequence of SEQ ID NO: 192; or        -   b) has an amino acid sequence that has at least 80% amino            acid identity with the amino acid sequence of SEQ ID NO:            192; or        -   c) has an amino acid sequence that has 2, or 1 amino acid(s)            difference with the amino acid sequence of SEQ ID NO: 192,            wherein            -   at position 2 the A has been changed into G;            -   at position 8 the T has been changed into S;            -   at position 9 the A has been changed into G; and/or            -   at position 10 the Y has been changed into F; or        -   d) has an amino acid sequence selected from any one of SEQ            ID NO's: 192-196.

Embodiment 17: A polypeptide according to embodiment 3, wherein theamino acid sequences of said CDR1, CDR2 and CDR3 are set forth in:

-   -   SEQ ID No: 141, 162 and 186, respectively; or    -   SEQ ID No: 141, 163 and 187, respectively; or    -   SEQ ID No: 141, 164 and 186, respectively; or    -   SEQ ID No: 141, 166 and 186, respectively; or    -   SEQ ID No: 141, 167 and 186, respectively; or    -   SEQ ID No: 141, 167 and 189, respectively; or    -   SEQ ID No: 141, 168 and 186, respectively; or    -   SEQ ID No: 141, 168 and 187, respectively; or    -   SEQ ID No: 141, 169 and 190, respectively; or    -   SEQ ID No: 141, 170 and 186, respectively; or    -   SEQ ID No: 141, 171 and 186, respectively; or    -   SEQ ID No: 141, 174 and 186, respectively; or    -   SEQ ID No: 141, 175 and 187, respectively; or    -   SEQ ID No: 142, 165 and 188, respectively; or    -   SEQ ID No: 142, 173 and 188, respectively; or    -   SEQ ID No: 143, 164 and 186, respectively; or    -   SEQ ID No: 144, 172 and 187, respectively; or    -   SEQ ID No: 145, 172 and 187, respectively; or    -   SEQ ID No: 141, 214 and 186, respectively; or    -   SEQ ID No: 141, 215 and 186, respectively; or    -   SEQ ID No: 141, 216 and 186, respectively; or    -   SEQ ID No: 141, 217 and 186, respectively; or    -   SEQ ID No: 141, 218 and 186, respectively; or    -   SEQ ID No: 141, 219 and 186, respectively; or    -   SEQ ID No: 141, 220 and 186, respectively; or    -   SEQ ID No: 213, 221 and 186, respectively; or    -   SEQ ID No: 213, 214 and 186, respectively.

Embodiment 18: A polypeptide according to embodiment 3, wherein theamino acid sequences of said CDR1, CDR2 and CD R3 are set forth in:

-   -   SEQ ID No: 146, 176 and 191, respectively; or    -   SEQ ID No: 146, 177 and 191, respectively.

Embodiment 19: A polypeptide according to embodiment 3, wherein theamino acid sequences of said CDR1, CDR2 and CD R3 are set forth in:

-   -   SEQ ID No: 147, 178 and 192, respectively; or    -   SEQ ID No: 147, 179 and 192, respectively; or    -   SEQ ID No: 147, 179 and 194, respectively; or    -   SEQ ID No: 148, 179 and 193, respectively; or    -   SEQ ID No: 149, 179 and 192, respectively; or    -   SEQ ID No: 149, 180 and 192, respectively; or    -   SEQ ID No: 149, 181 and 192, respectively; or    -   SEQ ID No: 149, 183 and 192, respectively; or    -   SEQ ID No: 149, 185 and 192, respectively; or    -   SEQ ID No: 150, 179 and 194, respectively; or    -   SEQ ID No: 150, 182 and 194, respectively; or    -   SEQ ID No: 151, 179 and 193, respectively; or    -   SEQ ID No: 151, 182 and 194, respectively; or    -   SEQ ID No: 151, 184 and 196, respectively; or    -   SEQ ID No: 152, 179 and 195, respectively; or    -   SEQ ID No: 153, 179 and 194, respectively; or    -   SEQ ID No: 154, 182 and 194, respectively; or    -   SEQ ID No: 155, 179 and 195, respectively; or    -   SEQ ID No: 156, 181 and 192, respectively; or    -   SEQ ID No: 157, 179 and 194, respectively; or    -   SEQ ID No: 158, 179 and 192, respectively; or    -   SEQ ID No: 159, 178 and 192, respectively; or    -   SEQ ID No: 160, 179 and 194, respectively; or    -   SEQ ID No: 161, 179 and 194, respectively.

Embodiment 20: A polypeptide according to embodiment 3, wherein theamino acid sequences of said CDR1, CDR2 and CD R3 are set forth in: SEQID NO's: 141, 164 and 186, respectively, or SEQ ID NO's: 141, 162 and186, respectively.

Embodiment 21: A polypeptide according to embodiment 3, wherein theamino acid sequences of said CDR1, CDR2 and CD R3 are set forth in: SEQID NO's: 213, 214 and 186 respectively, SEQ ID NO's: 213, 221 and 186respectively, or SEQ ID NO's: 141, 162 and 186 respectively.

Embodiment 22: A polypeptide according to any one of embodiments 2 to10, wherein said anti-CX3CR1 immunoglobulin single variable domain is aVHH domain comprising the sequence set forth in:

-   -   a) the amino acid sequence of SEQ ID NO: 3;    -   b) amino acid sequences that have at least 90% amino acid        identity with the amino acid sequences of SEQ ID NO: 3;    -   c) amino acid sequences that have 11, 10, 9, 8, 7, 6, 5, 4, 3,        2, or 1 amino acid difference with the amino acid sequences of        SEQ ID NO: 3; or    -   d) an amino acid sequence of any one of SEQ ID NO: 1-48, 121-140        or 222-224.

Embodiment 23: A polypeptide according to any one of embodiments 2 to10, wherein said anti-CX3CR1 immunoglobulin single variable domain is aVHH domain comprising the sequence set forth in:

-   -   a) the amino acid sequence of SEQ ID NO: 49;    -   b) an amino acid sequence that has at least 95% amino acid        identity with the amino acid sequences of SEQ ID NO: 49;    -   c) an amino acid sequence that has 5, 4, 3, 2, or 1 amino acid        difference with the amino acid sequences of SEQ ID NO: 49; or    -   d) an amino acid sequence of any one of SEQ ID NO: 49-52.

Embodiment 24: A polypeptide according to any one of embodiments 2 to10, wherein said anti-CX3CR1 immunoglobulin single variable domain is aVHH domain comprising the sequence set forth in:

-   -   a) the amino acid sequence of SEQ ID NO: 67;    -   b) an amino acid sequence that has at least 90% amino acid        identity with the amino acid sequences of SEQ ID NO: 67;    -   c) an amino acid sequence that has 12, 11, 10, 9, 8, 7, 6, 5, 4,        3, 2, or 1 amino acid difference with the amino acid sequences        of SEQ ID NO: 67; or    -   d) an amino acid sequence of any one of SEQ ID NO: 53-120.

Embodiment 25: A polypeptide according to embodiment 2, wherein saidanti-CX3CR1 immunoglobulin single variable domain comprises the sequenceset forth in SEQ ID NO: 1 or SEQ ID NO: 3.

Embodiment 26: A polypeptide according to embodiment 2, wherein saidanti-CX3CR1 immunoglobulin single variable domain comprises the sequenceset forth in any one of SEQ ID NO: 121-140 or SEQ ID NO: 222-224.

Embodiment 27: A polypeptide according to any of one of the embodimentsabove, which is humanized and/or optimized for stability, potency,manufacturability and/or similarity to human framework regions.

Embodiment 28: A polypeptide according to embodiment 27, which ishumanized and/or sequence optimized in one or more of the followingpositions (according to Kabat numbering): 1, 11, 14, 16, 74, 83, 108.

Embodiment 29: A polypeptide according to embodiment 28, comprising oneor more of the following mutations: E1D, S11L, A14P, E16G, A74S, K83R,Q108L.

Embodiment 30: A polypeptide according to any one of embodiments 3-29,in which:

-   -   i) FR1 is selected from SEQ ID NO's: 198-204;    -   ii) FR2 is selected from SEQ ID NO's: 205-208;    -   iii) FR3 is selected form SEQ ID NO's: 209-210; and/or    -   iv) FR4 is selected from SEQ ID NO's: 211-212.

Embodiment 31: A polypeptide according to any one of embodiments 3-30,which is humanized and/or sequence optimized in one or more of thefollowing positions (according to Kabat numbering): 52, 53.

Embodiment 32: A polypeptide according to embodiment 31, comprising oneor more of the following mutations: N52S, S53T.

Embodiment 33: A polypeptide according to any one of embodiments 3-32,in which CDR2 is selected from SEQ ID NO's: 214-221.

Embodiment 34: A polypeptide according to any one of embodiments 2-33,wherein said anti-CX3CR1 immunoglobulin single variable domain comprisesthe sequence set forth in any of SEQ ID NO's: 138-140 or 222-224.

Embodiment 35: A polypetide according to any one of embodiments 22 to26, wherein said VHH domain consists of any one of said amino acidsequences.

Embodiment 36: A polypeptide according to any one of embodiments 2 to35, wherein said immunoglobulin single variable domain cross-blocks thebinding of at least one of the immunoglobulin single variable domains ofSEQ ID NO's: 1-120, 121-140 and 222-224 to CX3CR1.

Embodiment 37: A polypeptide according to any one of embodiments 2 to35, wherein said immunoglobulin single variable domain is cross-blockedfrom binding to CX3CR1 by at least one of the amino acid sequences ofSEQ ID NO's: 1-120, 121-140 and 222-224.

Embodiment 38: A polypeptide according to any one of embodiments 2 to37, wherein the polypeptide further comprises a half-life extendingmoiety.

Embodiment 39: A polypeptide according to embodiment 38, wherein saidhalf-life extending moiety is covalently linked to said polypeptide andis selected from the group consisting of an albumin binding moiety, suchas an anti-albumin immunoglobulin domain, a transferrin binding moiety,such as an anti-transferrin immunoglobulin domain, a polyethylene glycolmolecule, a recombinant polyethylene glycol molecule, human serumalbumin, a fragment of human serum albumin, an albumin binding peptideor a Fc domain.

Embodiment 40: A polypeptide according to embodiment 38 or 39, whereinsaid half-life extending moiety consists of an anti-albuminimmunoglobulin single variable domain.

Embodiment 41: A polypeptide according to embodiment 40, wherein theimmunoglobulin single variable domain is selected from a VHH domain, ahumanized VHH domain, a camelized VH domain, a domain antibody, a singledomain antibody and/or “dAb”s.

Embodiment 42: A polypeptide according to embodiment 41, wherein theanti-albumin immunoglobulin single variable domain is selected from SEQID NO's: 230-232.

Embodiment 43: A polypeptide according to any one of embodiment 2 to 39,wherein said polypeptide is linked to an Fc portion (such as a human Fc,for example as set forth in SEQ ID NO: 252), optionally via a suitablelinker or hinge region.

Embodiment 44: A polypeptide according to any one of embodiments 2 to39, wherein said polypeptide is further linked to one or more constantdomains (for example, 2 or 3 constant domains that can be used as partof/to form an Fc portion), to an Fc portion and/or to one or moreantibody parts, fragments or domains that confer one or more effectorfunctions to the polypeptide of the invention and/or may confer theability to bind to one or more Fc receptors, optionally via a suitablelinker or hinge region.

Embodiment 45: A polypeptide according to any one of embodiments 2 to37, wherein said polypeptide further comprises a second immunoglobulinsingle variable domain, preferably a second anti-CX3CR1 immunoglobulinsingle variable domain.

Embodiment 46: A polypeptide according to embodiment 45, wherein saidfirst and said second immunoglobulin single variable domains arecovalently linked by a linker peptide.

Embodiment 47: A polypeptide according to embodiment 45 or 46, whereinsaid second immunoglobulin single variable domains essentially consistof four framework regions (FR1 to FR4) and three complementarydetermining regions (CDR1 to CDR3).

Embodiment 48: A polypeptide according to any one of embodiments 45 to47, wherein said first and said second immunoglobulin single variabledomains are antibody domains.

Embodiment 49: A polypeptide according to any one of embodiments 45 to48, wherein said first and second immunoglobulin single variable domainsare a VH, VL, VHH, camelized VH, or VHH that is optimized for stability,potency, manufacturability and/or similarity to human framework regions.

Embodiment 50: A polypeptide according to any one of embodiments 45 to49, wherein said CDR1 to CDR3 of said second immunoglobulin singlevariable domain are set forth in any one of embodiments 11 to 21.

Embodiment 51: A polypeptide according to any one of embodiments 45 to50, wherein said first and said second immunoglobulin single variabledomains comprise the same CDR3.

Embodiment 52: A polypeptide according to embodiment 51, wherein saidCDR3 is set forth in any one of embodiment 11 to 13.

Embodiment 53: A polypeptide according to any one of embodiments 45 to53, wherein said first and said second immunoglobulin single variabledomains comprise the same CDR1, CDR2 and CDR3.

Embodiment 54: A polypeptide according to embodiment 53, wherein saidCDR1 to CDR3 are set forth in any one of embodiments 11 to 21.

Embodiment 55: A polypeptide according to any one of embodiments 45 to54, wherein said first and said second immunoglobulin single variabledomains comprise the same VHH domain.

Embodiment 56: A The polypeptide according to any one of embodiments 45to 55, wherein said VHH domain is set forth in any one of embodiments 22to 37.

Embodiment 57: A polypeptide comprising a first immunoglobulin singlevariable domain comprising the CDR1, CDR2 and CDR3 set forth SEQ IDNO's: 141, 164 and 186 or SEQ ID NO's: 141, 162 and 186 and a secondimmunoglobulin single variable domain as set forth in any one ofembodiments 2 to 37.

Such a polypeptide may in particular be a polypeptide according to anyof embodiments 45 to 56.

Embodiment 58: A polypeptide according to embodiment 57, wherein saidfirst immunoglobulin single variable domain comprises the CDR1, CDR2 andCDR3 set forth in SEQ ID NO's: 213, 214 and 186, SEQ ID NO's: 213, 221and 186 or SEQ ID NO's: 141, 162 and 186.

Embodiment 59: A polypeptide according to embodiment 57 or 58, whereinsaid second immunoglobulin single variable domain comprises the CDR1,CDR2 and CDR3 set forth SEQ ID NO's: 141, 164 and 186 or SEQ ID NO's:141, 162 and 186.

Embodiment 60: A polypeptide according to embodiment 57 Or 58, whereinsaid second immunoglobulin single variable domain comprises the CDR1,CDR2 and CDR3 set forth in: SEQ ID NO's: 213, 214 and 186, SEQ ID NO's:213, 221 and 186 or SEQ ID NO's: 141, 162 and 186.

Embodiment 61: A polypeptide comprising a first immunoglobulin singlevariable domain, wherein said first immunoglobulin single variabledomain is a VHH domain comprising the sequence set forth in SEQ ID NO: 1or SEQ ID NO: 3 and a second immunoglobulin single variable domainaccording to any one of embodiments 2 to 37.

Such a polypeptide may in particular be a polypeptide according to anyof embodiments 45 to 60.

Embodiment 62: A polypeptide according to embodiment 61, wherein saidfirst immunoglobulin single variable domain is a VHH domain comprisingthe sequence set forth in any one of SEQ ID NO: 121-140 or 222-224.

Embodiment 63: A polypeptide according to embodiment 61 or 62, whereinsaid second immunoglobulin single variable domain is a VHH domaincomprising the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3.

Embodiment 64: A polypeptide according to embodiment 63, wherein saidsecond immunoglobulin single variable domain is a VHH domain comprisingthe sequence set forth in any one of SEQ ID NO: 121-140 or 222-224.

Embodiment 65: A polypeptide according to any one of embodiments 45 to64, wherein the polypeptide further comprises a half-life extendingmoiety.

Embodiment 66: A polypeptide according to embodiment 65, wherein saidhalf-life extending moiety is covalently linked to said polypeptide andis selected from the group consisting of an albumin binding moiety, suchas an anti-albumin immunoglobulin domain, a transferrin binding moiety,such as an anti-transferrin immunoglobulin domain, a polyethylene glycolmolecule, a recombinant polyethylene glycol molecule, human serumalbumin, a fragment of human serum albumin, an albumin binding peptideor a Fc domain.

Embodiment 67: A polypeptide according to embodiment 66, wherein saidhalf-life extending moiety consists of an anti-albumin immunoglobulinsingle variable domain.

Embodiment 68: A polypeptide according to embodiment 67, wherein theimmunoglobulin single variable domain is selected from a VHH domain, ahumanized VHH domain, a camelized VH domain, a domain antibody, a singledomain antibody and/or “dAb”s.

Embodiment 69: A polypeptide according to embodiment 68, wherein theanti-albumin immunoglobulin single variable domain is selected from SEQID NO's: 230-232.

Embodiment 70: A polypeptide according to any one of embodiments 45 to64, wherein said polypeptide is linked to an Fc portion (such as a humanFc, for example as set forth in SEQ ID NO: 252), optionally via asuitable linker or hinge region.

Embodiment 71: A polypeptide according to any one of embodiments 45 to66, wherein said polypeptide is further linked to one or more constantdomains (for example, 2 or 3 constant domains that can be used as partof/to form an Fc portion), to an Fc portion and/or to one or moreantibody parts, fragments or domains that confer one or more effectorfunctions to the polypeptide of the invention and/or may confer theability to bind to one or more Fc receptors, optionally via a suitablelinker or hinge region.

Embodiment 72: A polypeptide comprising the amino acid sequence of anyone of SEQ ID NO: 225-227.

Embodiment 73: A polypeptide comprising the amino acid sequence of anyone of SEQ ID NO: 249 or 277-281.

Embodiment 74: A polypeptide comprising the amino acid sequence of anyone of SEQ ID NO: 257-262.

Embodiment 75: A polypeptide comprising the amino acid sequence of anyone of SEQ ID NO: 253 or 254.

Embodiment 76: A polypeptide comprising the amino acid sequence of anyone of SEQ ID NO: 263 or 266.

Embodiment 77: A polypeptide comprising the amino acid sequence of anyone of SEQ ID NO: 267-276 and 282.

Embodiment 78: A nucleic acid molecule comprising a region encoding apolypeptide according to any one of embodiments 1 to 77.

Embodiment 79: An expression vector comprising a nucleic acid moleculeaccording to embodiment 78.

Embodiment 80: A host cell carrying an expression vector comprising anucleic acid molecule, said nucleic acid molecule comprising a regionencoding a polypeptide according to any one of embodiments 1 to 77,wherein said host cell is capable of expressing a polypeptide accordingto any one of embodiments 1 to 77, and wherein said host cell is aprokaryotic or a eukaryotic cell.

Embodiment 81: A pharmaceutical composition comprising (i) as the activeingredient, one or more polypeptides according to any one of embodiments1 to 77, and (ii) a pharmaceutically acceptable carrier, and optionally(iii) a diluent, excipient, adjuvant and/or stabilizer.

Embodiment 82: A method of manufacturing a polypeptide according to anyone of embodiments 1 to 77, comprising the steps of

-   -   culturing a host cell under conditions that allow expression of        a polypeptide according to any one of embodiments 1 to 77,        wherein said host cell carrying an expression vector comprising        a nucleic acid molecule, said nucleic acid molecule comprising a        region encoding a polypeptide according to any one of        embodiments 1 to 77, and wherein said host cell is a prokaryotic        or a eukaryotic cell;    -   recovering said polypeptide; and    -   purifying said polypeptide.

Embodiment 83: A method of using a polypeptide according to any one ofembodiments 1 to 77 for the treatment, prevention or alleviation of adisease, disorder or condition, in particular in a human being.

Embodiment 84: The method of embodiment 83, wherein said disease,disorder or condition is a CX3CR1-associated disease, disorder orcondition.

Embodiment 85: The method of embodiment 83, wherein said disease,disorder or condition is atherosclerosis.

Embodiment 86: An injectable pharmaceutical composition comprising thepolypeptide according to any one of embodiments 1 to 77, saidcomposition being suitable for intravenous or subcutaneous injection ina human being.

Embodiment 87: A method for preventing and/or treating a disease ordisorder that is associated with CX3CR1, wherein said method comprisesadministering to a subject in need thereof a pharmaceutically activeamount of at least one polypeptide according to any one of embodiments 1to 77.

Embodiment 88: A method of embodiment 85, further comprisingadministering an additional therapeutic agent selected from the groupconsisting of a statin, an antiplatelet, an anticoagulant, anantidiabetic and an antihypertensive.

Embodiment 89: A method for inhibiting the binding of CX3CR1 tofractalkine in a mammalian cell, comprising administering to the cell apolypeptide according to any one of embodiments 1 to 77, wherebysignaling mediated by the fractalkine is inhibited.

Embodiment 90: A method for detecting and/or quantifying CX3CR1 levelsin a biological sample by contacting the sample with a polypeptideaccording to any one of embodiments 1 to 77 and detecting binding of thepolypeptide with CX3CR1.

Embodiment 91: A method for diagnosing an CX3CR1-associated disorder orfor determining if a subject has an increased risk of developing anCX3CR1-associated disorder, wherein the method comprises contacting abiological sample from a subject with a polypeptide according to any oneof embodiments 1 to 77 and detecting binding of the polypeptide toCX3CR1 to determine the expression or concentration of CX3CR1.

Embodiment 92. A polypeptide according to any one of embodiments 1 to 77for use in the treatment, prevention or alleviation of a disease,disorder or condition, in a human being.

Embodiment 93. The polypeptide for use according to embodiment 92,wherein the disease, disorder or condition is a CX3CR1-associateddisease, disorder or condition.

Embodiment 94. The polypeptide for use according to embodiment 92,wherein the disease, disorder or condition is selected from cardio- andcerebrovascular atherosclerotic disorders, peripheral artery disease,restenosis, diabetic nephropathy, glomerulomephritis, human crescenticglomerulonephritis, IgA nephropathy, membranous nephropathy, lupusnephritis, vasculitis including Henoch-Schonlein purpura and Wegener'sgranulomatosis, rheumatoid arthritis, osteoarthritis, allograftrejection, systemic sclerosis, neurodegenerative disorders anddemyelinating disease, multiple sclerosis (MS), Alzheimer's disease,pulmonary diseases such as COPD, asthma,neuropathic pain, inflammatorypain, or cancer.

Embodiment 95. The polypeptide for use according to embodiment 92,wherein the disease, disorder or condition is atherosclerosis.

Embodiment 96. Use of a polypeptide according to any of embodiments 1 to77 for the manufacture of a medicament for the treatment, prevention oralleviation of a disease, disorder or condition, in a human being.

Embodiment 97. The method according to embodiment 87, wherein thedisease or disorder is selected from cardio- and cerebrovascularatherosclerotic disorders, peripheral artery disease, restenosis,diabetic nephropathy, glomerulonephritis, human crescenticglomerulonephritis, IgA nephropathy, membranous nephropathy, lupusnephritis, vasculitis including Henoch-Schonlein purpura and Wegener'sgranulomatosis, rheumatoid arthritis, osteoarthritis, allograftrejection, systemic sclerosis, neurodegenerative disorders anddemyelinating disease, multiple sclerosis (MS), Alzheimer's disease,pulmonary diseases such as COPD, asthma,neuropathic pain, inflammatorypain, or cancer.

Embodiment 98. The method according to embodiment 87, wherein thedisease, disorder or condition is atherosclerosis.

Embodiment 99. A diagnostic kit or diagnostic method comprising apolypeptide according to any one of embodiments 1 to 77, or the usethereof.

Embodiment 100. A diagnostic kit or diagnostic method according toembodiment 99, for the diagnosis of at least one of cardio- andcerebrovascular atherosclerotic disorders, peripheral artery disease,restenosis, diabetic nephropathy, glomerulomephritis, human crescenticglomerulonephritis, IgA nephropathy, membranous nephropathy, lupusnephritis, vasculitis including Henoch-Schonlein purpura and Wegener'sgranulomatosis, rheumatoid arthritis, osteoarthritis, allograftrejection, systemic sclerosis, neurodegenerative disorders anddemyelinating disease, multiple sclerosis (MS), Alzheimer's disease,pulmonary diseases such as COPD, asthma,neuropathic pain, inflammatorypain, or cancer.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The above and other aspects and embodiments of the invention will becomeclear from the further description herein, in which:

-   a) Unless indicated or defined otherwise, all terms used have their    usual meaning in the art, which will be clear to the skilled person.    Reference is for example made to the standard handbooks, such as    Sambrook et al, “Molecular Cloning: A Laboratory Manual” (2nd Ed.),    Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, “Genes    IV”, Oxford University Press, New York, (1990), and Roitt et al.,    “Immunology” (2^(nd) Ed.), Gower Medical Publishing, London, New    York (1989), as well as to the general background art cited herein;    Furthermore, unless indicated otherwise, all methods, steps,    techniques and manipulations that are not specifically described in    detail can be performed and have been performed in a manner known    per se, as will be clear to the skilled person. Reference is for    example again made to the standard handbooks, to the general    background art referred to above and to the further references cited    therein;-   b) Unless indicated otherwise, the terms “immunoglobulin” and    “immunoglobulin sequence”—whether used herein to refer to a heavy    chain antibody or to a conventional 4-chain antibody—are used as    general terms to include both the full-size antibody, the individual    chains thereof, as well as all parts, domains or fragments thereof    (including but not limited to antigen-binding domains or fragments    such as VHH domains or VH/VL domains, respectively). In addition,    the term “sequence” as used herein (for example in terms like    “immunoglobulin sequence”, “antibody sequence”, “(single) variable    domain sequence”, “VHH sequence” or “protein sequence”), should    generally be understood to include both the relevant amino acid    sequence as well as nucleic acid sequences or nucleotide sequences    encoding the same, unless the context requires a more limited    interpretation;-   c) The term “domain” (of a polypeptide or protein) as used herein    refers to a folded protein structure which has the ability to retain    its tertiary structure independently of the rest of the protein.    Generally, domains are responsible for discrete functional    properties of proteins, and in many cases may be added, removed or    transferred to other proteins without loss of function of the    remainder of the protein and/or of the domain.-   d) The term “immunoglobulin domain” as used herein refers to a    globular region of an antibody chain (such as e.g. a chain of a    conventional 4-chain antibody or of a heavy chain antibody), or to a    polypeptide that essentially consists of such a globular region.    Immunoglobulin domains are characterized in that they retain the    immunoglobulin fold characteristic of antibody molecules, which    consists of a 2-layer sandwich of about 7 antiparallel beta-strands    arranged in two beta-sheets, optionally stabilized by a conserved    disulphide bond.-   e) The term “immunoglobulin variable domain” as used herein means an    immunoglobulin domain essentially consisting of four “framework    regions” which are referred to in the art and hereinbelow as    “framework region 1” or “FR1”; as “framework region 2” or“FR2”; as    “framework region 3” or “FR3”; and as “framework region 4” or “FR4”,    respectively; which framework regions are interrupted by three    “complementarity determining regions” or “CDRs”, which are referred    to in the art and hereinbelow as “complementarity determining region    1”or “CDR1”; as “complementarity determining region 2” or “CDR2”;    and as “complementarity determining region 3” or “CDR3”,    respectively. Thus, the general structure or sequence of an    immunoglobulin variable domain can be indicated as follows:    FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulin variable    domain(s) that confer specificity to an antibody for the antigen by    carrying the antigen-binding site.-   f) The terms “immunoglobulin single variable domain” and “single    variable domain” as used herein mean an immunoglobulin variable    domain which is capable of specifically binding to an epitope of the    antigen without pairing with an additional variable immunoglobulin    domain. One example of immunoglobulin single variable domains in the    meaning of the present invention are “domain antibodies”, such as    the immunoglobulin single variable domains VH and VL (VH domains and    VL domains). Another example of immunoglobulin single variable    domains are “VHH domains” (or simply “VHHs”) from camelids, as    defined hereinafter.

In view of the above definition, the antigen-binding domain of aconventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgEmolecule; known in the art) or of a Fab fragment, a F(ab′)2 fragment, anFv fragment such as a disulphide linked Fv or a scFv fragment, or adiabody (all known in the art) derived from such conventional 4-chainantibody, would normally not be regarded as an immunoglobulin singlevariable domain, as, in these cases, binding to the respective epitopeof an antigen would normally not occur by one (single) immunoglobulindomain but by a pair of (associating) immunoglobulin domains such aslight and heavy chain variable domains, i.e. by a VH-VL pair ofimmunoglobulin domains, which jointly bind to an epitope of therespective antigen.

-   f1) “VHH domains”, also known as VHHs, V_(H)H domains, VHH antibody    fragments, and VHH antibodies, have originally been described as the    antigen binding immunoglobulin (variable) domain of “heavy chain    antibodies” (i.e. of “antibodies devoid of light chains”;    Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers    C, Songa E B, Bendahman N, Hamers R.: “Naturally occurring    antibodies devoid of light chains”; Nature 363, 446-448 (1993)). The    term “VHH domain” has been chosen in order to distinguish these    variable domains from the heavy chain variable domains that are    present in conventional 4-chain antibodies (which are referred to    herein as “V_(H) domains” or “VH domains”) and from the light chain    variable domains that are present in conventional 4-chain antibodies    (which are referred to herein as “V_(L) domains” or “VL domains”).    VHH domains can specifically bind to an epitope without an    additional antigen binding domain (as opposed to VH or VL domains in    a conventional 4-chain antibody, in which case the epitope is    recognized by a VL domain together with a VH domain). VHH domains    are small, robust and efficient antigen recognition units formed by    a single immunoglobulin domain.

In the context of the present invention, the terms VHH domain, VHH,V_(H)H domain, VHH antibody fragment, VHH antibody, as well as“Nanobody®” and “Nanobody® domain” (“Nanobody” being a trademark of thecompany Ablynx N. V.; Ghent; Belgium) are used interchangeably and arerepresentatives of immunoglobulin single variable domains (having thestructure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and specifically binding to anepitope without requiring the presence of a second immunoglobulinvariable domain), and which are distinguished from VH domains by theso-called “hallmark residues”, as defined in e.g. WO2009/109635, FIG. 1.

The amino acid residues of a VHH domain are numbered according to thegeneral numbering for V_(H) domains given by Kabat et al. (“Sequence ofproteins of immunological interest”, US Public Health Services, NIHBethesda, Md., Publication No. 91), as applied to VHH domains fromCamelids, as shown e.g. in FIG. 2 of Riechmann and Muyldermans, J.Immunol. Methods 231, 25-38 (1999). According to this numbering,

-   -   FR1 comprises the amino acid residues at positions 1-30,    -   CDR1 comprises the amino acid residues at positions 31-35,    -   FR2 comprises the amino acids at positions 36-49,    -   CDR2 comprises the amino acid residues at positions 50-65,    -   FR3 comprises the amino acid residues at positions 66-94,    -   CDR3 comprises the amino acid residues at positions 95-102, and    -   FR4 comprises the amino acid residues at positions 103-113.

However, it should be noted that—as is well known in the art for V_(H)domains and for VHH domains—the total number of amino acid residues ineach of the CDRs may vary and may not correspond to the total number ofamino acid residues indicated by the Kabat numbering (that is, one ormore positions according to the Kabat numbering may not be occupied inthe actual sequence, or the actual sequence may contain more amino acidresidues than the number allowed for by the Kabat numbering). This meansthat, generally, the numbering according to Kabat may or may notcorrespond to the actual numbering of the amino acid residues in theactual sequence.

Alternative methods for numbering the amino acid residues of V_(H)domains, which methods can also be applied in an analogous manner to VHHdomains, are known in the art. However, in the present description,claims and figures, the numbering according to Kabat and applied to VHHdomains as described above will be followed, unless indicated otherwise.

The total number of amino acid residues in a VHH domain will usually bein the range of from 110 to 120, often between 112 and 115. It shouldhowever be noted that smaller and longer sequences may also be suitablefor the purposes described herein.

Further structural characteristics and functional properties of VHHdomains and polypeptides containing the same can be summarized asfollows:

VHH domains (which have been “designed” by nature to functionally bindto an antigen without the presence of, and without any interaction with,a light chain variable domain) can function as a single, relativelysmall, functional antigen-binding structural unit, domain orpolypeptide. This distinguishes the VHH domains from the VH and VLdomains of conventional 4-chain antibodies, which by themselves aregenerally not suited for practical application as single antigen-bindingproteins or immunoglobulin single variable domains, but need to becombined in some form or another to provide a functional antigen-bindingunit (as in for example conventional antibody fragments such as Fabfragments; in scFv's, which consist of a VH domain covalently linked toa VL domain).

Because of these unique properties, the use of VHH domains—either aloneor as part of a larger polypeptide—offers a number of significantadvantages over the use of conventional VH and VL domains, scFv's orconventional antibody fragments (such as Fab- or F(ab′)2-fragments):

-   -   only a single domain is required to bind an antigen with high        affinity and with high selectivity, so that there is no need to        have two separate domains present, nor to assure that these two        domains are present in the right spacial conformation and        configuration (i.e. through the use of especially designed        linkers, as with scFv's);    -   VHH domains can be expressed from a single gene and require no        post-translational folding or modifications;    -   VHH domains can easily be engineered into multivalent and        multispecific formats (as further discussed herein);    -   VHH domains are highly soluble and do not have a tendency to        aggregate (as with the mouse-derived antigen-binding domains        described by Ward et al., Nature 341: 544-546 (1989));    -   VHH domains are highly stable to heat, pH, proteases and other        denaturing agents or conditions and, thus, may be prepared,        stored or transported without the use of refrigeration        equipments, conveying a cost, time and environmental savings;    -   VHH domains are easy and relatively cheap to prepare, even on a        scale required for production. For example, VHH domains and        polypeptides containing the same can be produced using microbial        fermentation (e.g. as further described below) and do not        require the use of mammalian expression systems, as with for        example conventional antibody fragments;    -   VHH domains are relatively small (approximately 15 kDa, or 10        times smaller than a conventional IgG) compared to conventional        4-chain antibodies and antigen-binding fragments thereof, and        therefore    -   show high(er) penetration into tissues and    -   can be administered in higher doses than such conventional        4-chain antibodies and antigen-binding fragments thereof;    -   VHH domains can show so-called cavity-binding properties (inter        alia due to their extended CDR3 loop, compared to conventional        VH domains) and can therefore also access targets and epitopes        not accessible to conventional 4-chain antibodies and        antigen-binding fragments thereof.

Methods of obtaining VHH domains binding to a specific antigen orepitope have been described earlier, e.g. in WO2006/040153 andWO2006/122786. As also described therein in detail, VHH domains derivedfrom camelids can be “humanized” by replacing one or more amino acidresidues in the amino acid sequence of the original VHH sequence by oneor more of the amino acid residues that occur at the correspondingposition(s) in a VH domain from a conventional 4-chain antibody from ahuman being. A humanized VHH domain can contain one or more fully humanframework region sequences, and, in an even more specific embodiment,can contain human framework region sequences derived from DP-29, DP-47,DP-51, or parts thereof, optionally combined with JH sequences, such asJH5.

-   f2) “Domain antibodies”, also known as “Dab”s, “Domain Antibodies”,    and “dAbs” (the terms “Domain Antibodies” and “dAbs” being used as    trademarks by the GlaxoSmithKline group of companies) have been    described in e.g. Ward, E. S., et al.: “Binding activities of a    repertoire of single immunoglobulin variable domains secreted from    Escherichia coli”; Nature 341: 544-546 (1989); Holt, L. J. et al.:    “Domain antibodies: proteins for therapy”; TRENDS in Biotechnology    21(11): 484-490 (2003); and WO2003/002609.

Domain antibodies essentially correspond to the VH or VL domains ofnon-camelid mammalians, in particular human 4-chain antibodies. In orderto bind an epitope as a single antigen binding domain, i.e. withoutbeing paired with a VL or VH domain, respectively, specific selectionfor such antigen binding properties is required, e.g. by using librariesof human single VH or VL domain sequences. Domain antibodies have, likeVHHs, a molecular weight of approximately 13 to approximately 16 kDaand, if derived from fully human sequences, do not require humanizationfor e.g. therapeutical use in humans. As in the case of VHH domains,they are well expressed also in prokaryotic expression systems,providing a significant reduction in overall manufacturing cost.

Domain antibodies, as well as VHH domains, can be subjected to affinitymaturation by introducing one or more alterations in the amino acidsequence of one or more CDRs, which alterations result in an improvedaffinity of the resulting immunoglobulin single variable domain for itsrespective antigen, as compared to the respective parent molecule.Affinity-matured immunoglobulin single variable domain molecules of theinvention may be prepared by methods known in the art, for example, asdescribed by Marks et al., 1992, Biotechnology 10:779-783, or Barbas, etal., 1994, Proc. Nat. Acad. Sci, USA 91: 3809-3813.; Shier et al., 1995,Gene 169:147-155; Yelton et al., 1995, Immunol. 155: 1994-2004; Jacksonet al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J.Mol. Biol. 226(3): 889 896; KS Johnson and RE Hawkins, “Affinitymaturation of antibodies using phage display”, Oxford University Press1996.

-   f3) Furthermore, it will also be clear to the skilled person that it    is possible to “graft” one or more of the CDR's mentioned above onto    other “scaffolds”, including but not limited to human scaffolds or    non-immunoglobulin scaffolds. Suitable scaffolds and techniques for    such CDR grafting are known in the art.-   g) The terms “epitope” and “antigenic determinant”, which can be    used interchangeably, refer to the part of a macromolecule, such as    a polypeptide, that is recognized by antigen-binding molecules, such    as conventional antibodies or the polypeptides of the invention, and    more particularly by the antigen-binding site of said molecules.    Epitopes define the minimum binding site for an immunoglobulin, and    thus represent the target of specificity of an immunoglobulin.

The part of an antigen-binding molecule (such as a conventional antibodyor a polypeptide of the invention) that recognizes the epitope is calleda paratope.

-   h) The term “biparatopic” (antigen-)binding molecule or    “biparatopic” polypeptide as used herein shall mean a polypeptide    comprising a first immunoglobulin single variable domain and a    second immunoglobulin single variable domain as herein defined,    wherein these two variable domains are capable of binding to two    different epitopes of one antigen, which epitopes are not normally    bound at the same time by one monospecific immunoglobulin, such as    e.g. a conventional antibody or one immunoglobulin single variable    domain. Biparatopic polypeptides can be composed of variable domains    which have different epitope specificities, and do not contain    mutually complementary variable domain pairs which bind to the same    epitope. The two variable domains do therefore not compete with each    other for binding to the target.-   i) A polypeptide (such as an immunoglobulin, an antibody, an    immunoglobulin single variable domain, a polypeptide of the    invention, or generally an antigen binding molecule or a fragment    thereof) that can “bind to” or “specifically bind to”, that “has    affinity for” and/or that “has specificity for” a certain epitope,    antigen or protein (or for at least one part, fragment or epitope    thereof) is said to be “against” or “directed against” said epitope,    antigen or protein or is a “binding” molecule with respect to such    epitope, antigen or protein, or is said to be “anti”-epitope,    “anti”-antigen or “anti”-protein (e.g anti-CX3CR1).-   k) Generally, the term “specificity” refers to the number of    different types of antigens or epitopes to which a particular    antigen-binding molecule or antigen-binding protein (such as an    immunoglobulin, an antibody, an immunoglobulin single variable    domain, or a polypeptide of the invention) can bind. The specificity    of an antigen-binding protein can be determined based on its    affinity and/or avidity. The affinity, represented by the    equilibrium constant for the dissociation of an antigen with an    antigen-binding protein (KD), is a measure for the binding strength    between an epitope and an antigen-binding site on the    antigen-binding protein: the lesser the value of the KD, the    stronger the binding strength between an epitope and the    antigen-binding molecule (alternatively, the affinity can also be    expressed as the affinity constant (KA), which is 1/KD). As will be    clear to the skilled person (for example on the basis of the further    disclosure herein), affinity can be determined in a manner known per    se, depending on the specific antigen of interest. Avidity is the    measure of the strength of binding between an antigen-binding    molecule (such as an immunoglobulin, an antibody, an immunoglobulin    single variable domain, or a polypeptide of the invention) and the    pertinent antigen. Avidity is related to both the affinity between    an epitope and its antigen binding site on the antigen-binding    molecule and the number of pertinent binding sites present on the    antigen-binding molecule.-   l) Amino acid residues will be indicated according to the standard    three-letter or one-letter amino acid code, as generally known and    agreed upon in the art. When comparing two amino acid sequences, the    term “amino acid difference” refers to insertions, deletions or    substitutions of the indicated number of amino acid residues at a    position of the reference sequence, compared to a second sequence.    In case of substitution(s), such substitution(s) will preferably be    conservative amino acid substitution(s), which means that an amino    acid residue is replaced with another amino acid residue of similar    chemical structure and which has little or essentially no influence    on the function, activity or other biological properties of the    polypeptide. Such conservative amino acid substitutions are well    known in the art, for example from WO 98/49185, wherein conservative    amino acid substitutions preferably are substitutions in which one    amino acid within the following groups (i)-(v) is substituted by    another amino acid residue within the same group: (i) small    aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro    and Gly; (ii) polar, negatively charged residues and their    (uncharged) amides: Asp, Asn, Glu and Gln; (iii) polar, positively    charged residues: His, Arg and Lys; (iv) large aliphatic, nonpolar    residues: Met, Leu, Ile, Val and Cys; and (v) aromatic residues:    Phe, Tyr and Trp. Particularly preferred conservative amino acid    substitutions are as follows:-   Ala into Gly or into Ser;-   Arg into Lys;-   Asn into Gln or into His;-   Asp into Glu;-   Cys into Ser;-   Gln into Asn;-   Glu into Asp;-   Gly into Ala or into Pro;-   His into Asn or into Gln;-   Ile into Leu or into Val;-   Leu into Ile or into Val;-   Lys into Arg, into Gln or into Glu;-   Met into Leu, into Tyr or into Ile;-   Phe into Met, into Leu or into Tyr;-   Ser into Thr;-   Thr into Ser;-   Trp into Tyr;-   Tyr into Trp or into Phe;-   Val into Ile or into Leu.-   m) A nucleic acid or polypeptide molecule is considered to be “(in)    essentially isolated (form)”—for example, when compared to its    native biological source and/or the reaction medium or cultivation    medium from which it has been obtained—when it has been separated    from at least one other component with which it is usually    associated in said source or medium, such as another nucleic acid,    another protein/polypeptide, another biological component or    macromolecule or at least one contaminant, impurity or minor    component. In particular, a nucleic acid or polypeptide molecule is    considered “essentially isolated” when it has been purified at least    2-fold, in particular at least 10- fold, more in particular at least    100-fold, and up to 1000-fold or more. A nucleic acid or polypeptide    molecule that is “in essentially isolated form” is preferably    essentially homogeneous, as determined using a suitable technique,    such as a suitable chromatographical technique, such as    polyacrylamide-gelelectrophoresis;-   n) “Sequence identity” between e.g. two immunoglobulin single    variable domain sequences indicates the percentage of amino acids    that are identical between these two sequences. It may be calculated    or determined as described in paragraph f) on pages 49 and 50 of    WO08/020079. “Sequence similarity” indicates the percentage of amino    acids that either are identical or that represent conservative amino    acid substitutions.

Target Specificity

The polypeptides of the invention have specificity for human CX3CR1.Thus, the polypeptides of the invention preferably bind to human CX3CR1(SEQ ID NO: 255). In one aspect, the polypeptides of the presentinvention also bind to cynomolgus CX3CR1 (SEQ ID NO: 256).

Polypeptides of the Invention

The invention provides novel pharmaceutically active agents for theprevention, treatment, alleviation and/or diagnosis of CX3CR1 associateddiseases, disorders or conditions, such as cardiovascular diseases. Inparticular, the invention provides polypeptides which bind to humanCX3CR1 and are capable of blocking the binding of human fractalkine tohuman CX3CR1. In one aspect, the polypeptide is an immunoglobulincomprising an antigen-binding domain comprising three complementaritydetermining regions CDR1, CDR2 and CDR3, wherein said immunoglobulinbinds to human CX3CR1 and is capable of blocking the binding of humanfractalkine to human CX3CR1. In a further aspect, the polypeptidecomprises one or more anti-CX3CR1 immunoglobulin single variable domain,wherein said polypeptide is capable of blocking the binding of humanfractalkine to human CX3CR1.

In one aspect, a polypeptide of the present invention is characterizedby one or more of the following properties:

-   -   Bind with high affinity to human CX3CR1, for example at an EC₅₀        of less than or equal to 10 nM, less than or equal to 5 nM, less        than or equal to 2.5 nM or less than or equal to 1 nM, as        determined by cell binding FACS;    -   Block the binding of human fractalkine to human CX3CR1, for        example at an IC50 of less than or equal to 300 nM, or less than        or equal to 100 nM, or less than or equal to 20 nM, or less than        or equal to 10 nM, or less than or equal to 5 nM, or less than        or equal to 2.5 nM or less than or equal to 1 nM;    -   Inhibit fractalkine induced chemotaxis mediated by human CX3CR1,        for example at an IC₅₀ of less than or equal to 500 nM, or less        than or equal to 100 nM, or of less than or equal to 75 nM, or        less than or equal to 50 nM, or less than or equal to 10 nM or        less than or equal to 5 nM; the obtained efficacy of inhibition        is more than or equal to 15%, or more than or equal to 50%, or        more than or equal to 80%, or more than or equal to 95%;    -   Inhibit fractalkine induced internalization mediated by human        CX3CR1, for example at an IC₅₀ of less than or equal to 10 nM or        less than or equal to 5 nM;    -   Cross-react with cynomolgus CX3CR1, for example within 10-fold        of E/IC₅₀ for human CX3CR1 for binding and functional        inhibition.

In a further aspect, a polypeptide of the present invention furthercomprises a half-life extending moiety, for example an albumin bindingmoiety, a polyethylene glycol molecule or a Fc domain. In a furtheraspect, a polypeptide of the present invention comprises two or moreanti-CX3CR1 immunoglobulin single variable domains. In one aspect, thetwo anti-CX3CR1 immunoglobulin single variable domains are covalentlylinked by a linker peptide. In one aspect, the two anti-CX3CR1immunoglobulin single variable domains in a polypeptide of the presentinvention have the same amino acid sequence. In another aspect, the twoanti-CX3CR1 immunoglobulin single variable domains in a polypeptide ofthe present invention have different amino acid sequences. In oneaspect, a polypeptide of the present invention comprises two anti-CX3CR1immunoglobulin single variable domains and further comprises a half-lifeextending moiety, for example an albumin binding moiety, a polyethyleneglycol molecule or a Fc domain. In one aspect, a polypeptide of thepresent invention comprises a first anti-CX3CR1 immunoglobulin singlevariable domain covalently linked to an albumin binding moiety by afirst linker peptide, wherein said albumin binding moiety is furthercovalently linked to a second anti-CX3CR1 immunoglobulin single variabledomain by a second linker peptide.

In one aspect, a polypeptide of the present invention comprises ananti-CX3CR1 immunoglobulin single variable domain covalently linked to aFc domain by a linker peptide. In one aspect, such polypeptidecomprising an anti-CX3CR1 immunoglobulin single variable domaincovalently linked to a Fc domain by a linker peptide is provided as adimer, for example through disulfide bridges.

Polypeptides according to the present invention are obtained asdescribed hereinbelow. In summary, single variable domains of thepresent invention were identified from a library expressing singlevariable domains (VHH) derived from a Ilama immunized with DNA encodinghuman CX3CR1. The phage library was panned on hCX3CR1 virallipoparticles and binding phage were screened for their ability tocompete for receptor binding with Alexa-fluor labeled fractalkine(AF-FKN). Representative single variable domains of the presentinvention are described herein in further details.

In one aspect, an immunoglobulin single variable domain of the presentinvention consists essentially of four framework regions (FR1, FR2, FR3and FR4) and three complementary determining regions (CDR1, CDR2 andCDR3). In particular, the immunoglobulin single variable domain has thestructure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In one aspect, theimmunoglobulin single variable domain is an antibody domain.

In one aspect, the CDR3 of a polypeptide of the present invention, inparticular a immunoglobulin single domain of the present invention hasthe amino acid sequence of Asp-Xaa1-Arg-Arg-Gly-Trp-Xaa2-Xaa3-Xaa4-Xaa5as set forth in SEQ ID NO: 197, wherein:

-   -   Xaa1 is Pro, Ala or Gly;    -   Xaa2 is Asp or Asn;    -   Xaa3 is Thr or Ser;    -   Xaa4 is Arg, Lys, Ala or Gly; and    -   Xaa5 is Tyr or Phe.

In one aspect, the CDR3 of a polypeptide of the present invention, inparticular a immunoglobulin single domain of the present invention, hasthe amino acid sequence of Asp-Xaa1-Arg-Arg-Gly-Trp-Xaa2-Xaa3-Xaa4-Xaa5as set forth in SEQ ID NO: 197, wherein:

-   -   Xaa1 is Pro, Ala or Gly;    -   Xaa2 is Asp or Asn;    -   Xaa3 is Thr;    -   Xaa4 is Arg or Lys; and    -   Xaa5 is Tyr.

In one aspect, the CDR3 of a polypeptide of the present invention, inparticular an immunoglobuling single domain of the present invention,has the amino acid sequence of Asp-Pro-Arg-Arg-Gly-Trp-Asp-Thr-Arg-Tyras set forth in SEQ ID NO: 186.

In a further aspect, a polypeptide of the present invention, inparticular an immunoglobuling single domain of the present invention,has the following CDR1, CDR2 and CDR3:

-   -   CDR1:        -   a) has the amino acid sequence of GSIFSSNAMA (SEQ ID NO:            141); or        -   b) has an amino acid sequence that has at least 80% amino            acid identity with the amino acid sequence of SEQ ID NO:            141; or        -   c) has an amino acid sequence that has 2, or 1 amino acid(s)            difference with the amino acid sequence of SEQ ID NO: 141,            wherein            -   at position 2 the S has been changed into T, or G;            -   at position 6 the S has been changed into R;            -   at position 7 the N has been changed into T; and/or            -   at position 9 the M has been changed into K; or        -   d) has an amino acid sequence selected from any one of SEQ            ID NO's: 141-145 and 213;    -   CDR2:        -   a) has the amino acid sequence of GINSVGITK (SEQ ID NO:            164); or        -   b) has an amino acid sequence that has at least 70% amino            acid identity with the amino acid sequence of SEQ ID NO:            164; or        -   c) has an amino acid sequence that has 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 164, wherein            -   at position 1 the G has been changed into A, L, V or S;            -   at position 3 the N has been changed into D, S, Q, G or                T;            -   at position 4 the S has been changed into T, K, G or P;            -   at position 5 the V has been changed into A;            -   at position 6 the G has been changed into D;            -   at position 7 the I has been changed into T, or V;            -   at position 8 the T has been changed into A; and/or            -   at position 9 the K has been changed into R; or        -   d) has an amino acid sequence selected from any one of SEQ            ID NO's: 162-175 and 214-221; and    -   CDR3:        -   a) has the amino acid sequence of DPRRGWDTRY (SEQ ID NO:            186); or        -   b) has an amino acid sequence that has at least 70% amino            acid identity the amino acid sequence of SEQ ID NO: 186; or        -   c) has an amino acid sequence that has 3, 2, or 1 amino            acid(s) difference with the amino acid sequences of SEQ ID            NO: 186, wherein            -   at position 2 the P has been changed into A, or G;            -   at position 7 the D has been changed into N; and/or            -   at position 9 the R has been changed into K; or        -   d) has an amino acid sequence selected from any one of SEQ            ID NO's: 186-190.

In a further aspect, a polypeptide of the present invention, inparticular a immunoglobuling single domain of the present invention, hasthe following CDR1, CDR2 and CDR3, wherein:

-   -   said CDR1 has the amino acid sequence of GRTFSSYAMG (SEQ ID NO:        146);    -   said CDR2 has an amino acid sequence that a) has at least 90%        amino acid identity with the amino acid sequence of GISGSASRKY        (SEQ ID NO: 176) or b) has the amino acid sequence of SEQ ID NO:        176 or 177; and    -   said CDR3 has the amino acid sequence of SNSYPKVQFDY (SEQ ID NO:        191).

In a further aspect, a polypeptide of the present invention, inparticular an immunoglobuling single domain of the present invention,has the following CDR1, CDR2 and CDR3:

-   -   said CDR1:        -   a) has the amino acid sequence of GTIFSNNAMG (SEQ ID NO:            147); or        -   b) has an amino acid sequence that has 6, 5, 4, 3, 2, or 1            amino acid(s) difference with the amino acid sequence of SEQ            ID NO: 147, wherein            -   at position 1 the G has been changed into K, R, or A;            -   at position 2 the T has been changed into I, P, S or L;            -   at position 3 the I has been changed into V, or T;            -   at position 4 the F has been changed into L;            -   at position 5 the S has been changed into R, or D;            -   at position 6 the N has been changed into S, T, or D;                and/or            -   at position 7 the N has been changed into T, or Y; or        -   c) has an amino acid sequence selected from any one of SEQ            ID NO's: 147-161;    -   said CDR2:        -   a) has the amino acid sequence of SISNSGSTN (SEQ ID NO:            179); or        -   b) has an amino acid sequences that has 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 179, wherein            -   at position 3 the S has been changed into T, or G;            -   at position 4 the N has been changed into S, or I;            -   at position 5 the S has been changed into T;            -   at position 6 the G has been changed into Y; and/or            -   at position 8 the T has been changed into A; or        -   c) has an amino acid sequence selected from any one of SEQ            ID NO's: 178-185; and    -   said CDR3:        -   a) has the amino acid sequence of DARRGWNTAY (SEQ ID NO:            192); or        -   b) has an amino acid sequence that has at least 80% amino            acid identity with the amino acid sequence of SEQ ID NO:            192; or        -   c) has an amino acid sequence that has 2, or 1 amino acid(s)            difference with the amino acid sequence of SEQ ID NO: 192,            wherein            -   at position 2 the A has been changed into G;            -   at position 8 the T has been changed into S;            -   at position 9 the A has been changed into G; and/or            -   at position 10 the Y has been changed into F; or        -   d) has an amino acid sequence selected from any one of SEQ            ID NO's: 192-196.

In a further aspect, a polypeptide of the present invention, inparticular an immunoglobuling single domain of the present invention,has the following CDR1, CDR2 and CDR3:

-   -   SEQ ID No: 141, 162 and 186, respectively; or    -   SEQ ID No: 141, 163 and 187, respectively; or    -   SEQ ID No: 141, 164 and 186, respectively; or    -   SEQ ID No: 141, 166 and 186, respectively; or    -   SEQ ID No: 141, 167 and 186, respectively; or    -   SEQ ID No: 141, 167 and 189, respectively; or    -   SEQ ID No: 141, 168 and 186, respectively; or    -   SEQ ID No: 141, 168 and 187, respectively; or    -   SEQ ID No: 141, 169 and 190, respectively; or    -   SEQ ID No: 141, 170 and 186, respectively; or    -   SEQ ID No: 141, 171 and 186, respectively; or    -   SEQ ID No: 141, 174 and 186, respectively; or    -   SEQ ID No: 141, 175 and 187, respectively; or    -   SEQ ID No: 142, 165 and 188, respectively; or    -   SEQ ID No: 142, 173 and 188, respectively; or    -   SEQ ID No: 143, 164 and 186, respectively; or    -   SEQ ID No: 144, 172 and 187, respectively; or    -   SEQ ID No: 145, 172 and 187, respectively; or    -   SEQ ID No: 141, 214 and 186, respectively; or    -   SEQ ID No: 141, 215 and 186, respectively; or    -   SEQ ID No: 141, 216 and 186, respectively; or    -   SEQ ID No: 141, 217 and 186, respectively; or    -   SEQ ID No: 141, 218 and 186, respectively; or    -   SEQ ID No: 141, 219 and 186, respectively; or    -   SEQ ID No: 141, 220 and 186, respectively; or    -   SEQ ID No: 213, 221 and 186, respectively; or    -   SEQ ID No: 213, 214 and 186, respectively.

In a further aspect, a polypeptide of the present invention, inparticular an immunoglobuling single domain of the present invention,has the following CDR1, CDR2 and CDR3:

-   -   SEQ ID No: 146, 176 and 191, respectively; or    -   SEQ ID No: 146, 177 and 191, respectively.

In a further aspect, a polypeptide of the present invention, inparticular an immunoglobuling single domain of the present invention,has the following CDR1, CDR2 and CDR3:

-   -   SEQ ID No: 147, 178 and 192, respectively; or    -   SEQ ID No: 147, 179 and 192, respectively; or    -   SEQ ID No: 147, 179 and 194, respectively; or    -   SEQ ID No: 148, 179 and 193, respectively; or    -   SEQ ID No: 149, 179 and 192, respectively; or    -   SEQ ID No: 149, 180 and 192, respectively; or    -   SEQ ID No: 149, 181 and 192, respectively; or    -   SEQ ID No: 149, 183 and 192, respectively; or    -   SEQ ID No: 149, 185 and 192, respectively; or    -   SEQ ID No: 150, 179 and 194, respectively; or    -   SEQ ID No: 150, 182 and 194, respectively; or    -   SEQ ID No: 151, 179 and 193, respectively; or    -   SEQ ID No: 151, 182 and 194, respectively; or    -   SEQ ID No: 151, 184 and 196, respectively; or    -   SEQ ID No: 152, 179 and 195, respectively; or    -   SEQ ID No: 153, 179 and 194, respectively; or    -   SEQ ID No: 154, 182 and 194, respectively; or    -   SEQ ID No: 155, 179 and 195, respectively; or    -   SEQ ID No: 156, 181 and 192, respectively; or    -   SEQ ID No: 157, 179 and 194, respectively; or    -   SEQ ID No: 158, 179 and 192, respectively; or    -   SEQ ID No: 159, 178 and 192, respectively; or    -   SEQ ID No: 160, 179 and 194, respectively; or    -   SEQ ID No: 161, 179 and 194, respectively.

In a further aspect, a polypeptide of the present invention, inparticular an immunoglobuling single domain of the present invention,has the CDR1, CDR2 and CDR3 set forth in:

-   -   SEQ ID NO's: 141, 164 and 186; or    -   SEQ ID NO's: 141, 162 and 186.

In a further aspect, a polypeptide of the present invention, inparticular an immunoglobuling single domain of the present invention,has the CDR1, CDR2 and CDR3 set forth in:

-   -   SEQ ID NO's: 213, 214 and 186; or    -   SEQ ID NO's: 213, 221 and 186; or    -   SEQ ID NO's: 141, 162 and 186.

Representative polypeptides of the present invention having the CDRsdescribed above are shown in Tables 1, 2, 3 (representative polypeptidesof families 101, 9 and 13, respectively) and 4 (representativepolypeptides of optimized variants of family 101.

TABLE 1 Family 101 SEQ SEQ SEQ Nanobody SEQ CDR1* CDR1 CDR2* CDR2 CDR3*CDR3 CX3CR1BII  1 GSIFSSNAMA 141 AINSVGVTK 162 DPRRGWDTRY 186 PMP66B02CX3CR1BII  2 GSIFSSNAMA 141 VINSVGITK 163 DARRGWDTRY 187 PMP54Al2CX3CR1BII 3 GSIFSSNAMA 141 GINSVGITK 164 DPRRGWDTRY 186 PMP54A3CX3CR1BII  4 GSIFSSNAMA 141 GINSVGITK 164 DPRRGWDTRY 186 PMP54A4CX3CR1BII  5 GSIFSSNAMA 141 GINSVGITK 164 DPRRGWDTRY 186 PMP54A5CX3CR1BII  6 GTIFSSNAMA 142 GINSVDITK 165 DPRRGWNTRY 188 PMP54A7CX3CR1BII  7 GSIFSSNAMA 141 GINSVGITK 164 DPRRGWDTRY 186 PMP54B1CX3CR1BII  8 GTIFSSNAMA 142 GINSVDITK 165 DPRRGWNTRY 188 PMP54B2CX3CR1BII  9 GSIFSSNAMA 141 AINSVGITK 166 DPRRGWDTRY 186 PMP54B3CX3CR1BII 10 GSIFSSNAMA 141 GINSVGITK 164 DPRRGWDTRY 186 PMP54B5CX3CR1BII 11 GSIFSSNAMA 141 LINSVGITK 167 DGRRGWDTRY 189 PMP54D5CX3CR1BII 12 GSIFSSNAMA 141 GINSVGIKT 164 DPRRGWDTRY 186 PMP54D8CX3CR1BII 13 GSIFSSNAMA 141 AINSVGITK 166 DPRRGWDTRY 186 PMP54F6CX3CR1BII 14 GSIFSSNAMA 141 LINSVGITK 167 DPRRGWDTRY 186 PMP54G3CX3CR1BII 15 GTIFSSNAMA 142 GINSVDITK 165 DPRRGWNTRY 188 PMP54H1CX3CR1BII 16 GSIFSSNAMA 141 VINSVGITK 163 DARRGWDTRY 187 PMP54H4CX3CR1BII 17 GTIFSSNAMA 142 GINSVDITK 165 DPRRGWNTRY 188 PMP61F10CX3CR1BII 18 GSIFSSNAMA 141 LINSVGITK 167 DPRRGWDTRY 186 PMP61D1CX3CR1BII 19 GSIFSSNAMA 141 LINSVGITK 167 DPRRGWDTRY 186 PMP61D5CX3CR1BII 20 GSIFSSNAMA 141 GINSVGITK 164 DPRRGWDTRY 186 PMP61E2CX3CR1BII 21 GSIFSSNAMA 141 AINSVGITK 166 DPRRGWDTRY 186 PMP61F11CX3CR1BII 22 GSIFSSNAMA 141 LINSVGITK 167 DPRRGWDTRY 186 PMP61G2CX3CR1BII  23 GSIFSSNAMA 141 AINSVGITK 166 DPRRGWDTRY 186 PMP61G3CX3CR1BII  24 GSIFSSNAMA 141 AINSVGITK 166 DPRRGWDTRY 186 PMP61G4CX3CR1BII  25 GSIFSSNAMA 141 VINTVGITK 168 DARRGWDTRY 187 PMP61F4CX3CR1BII  26 GSIFSSNAMA 141 VINSVGITK 163 DARRGWDTRY 187 PMP61A11CX3CR1BII  27 GSIFSSNAMA 141 VINTVGITK 168 DARRGWDTRY 187 PMP61B2CX3CR1BII  28 GSIFSSNAMA 141 LIDSAGITK   169 DARRGWNTKY 190 PMP61C9CX3CR1BII  29 GSIFSSNAMA 141 AINSVGITK 166 DPRRGWDTRY 186 PMP65H02CX3CR1BII  30 GSIFSSNAMA 141 GINSVGIAK 170 DPRRGWDTRY 186 PMP65E11CX3CR1BII  31 GSIFSSNAKA 143 GINSVGITK 164 DPRRGWDTRY 186 PMP65E10CX3CR1BII  32 GSIFSSNAMA 141 GINSVGITK 164 DPRRGWDTRY 186 PMP65E05CX3CR1BII  33 GSIFSSNAMA 141 VINKVGITK 171 DPRRGWDTRY 186 PMP65B11CX3CR1BII  34 GSIFSSNAMA 141 AINSVGITK 166 DPRRGWDTRY 186 PMP65B07CX3CR1BII  35 GSIFSRNAMA 144 SINSVGITK 172 DARRGWDTRY 187 PMP65B09CX3CR1BII  36 GGIFSRNAMA 145 SINSVGITK 172 DARRGWDTRY 187 PMP65H01CX3CR1BII  37 GTIFSSNAMA 142 GINSVDITR 173 DPRRGWNTRY 188 PMP65G07CX3CR1BII  38 GSIFSSNMAA 141 LINSVGITK 167 DPRRGWDTRY 186 PMP66H08CX3CR1BII  39 GSIFSSNAMA 141 AINSVGITK 166 DPRRGWDTRY 186 PMP66H04CX3CR1BII 40 GSIFSSNAMA 141 LINSVGITK 167 DPRRGWDTRY 186 PMP66F02CX3CR1BII 41 GSIFSSNAMA 141 AINSVGTTK 174 DPRRGWDTRY 186 PMP66E11CX3CR1BII 42 GSIFSSNAMA 141 LINSVGITK 167 DPRRGWDTRY 186 PMP66D10CX3CR1BII 43 GSIFSSNAMA 141 GINSVGITK 164 DPRRGWDTRY 186 PMP66D08CX3CR1BII 44 GSIFSSNAMA 141 LINSVGITK 167 DPRRGWDTRY 186 PMP66A04CX3CR1BII 45 GTIFSSNAMA 142 GINSVDITK 165 DPRRGWNTRY 188 PMP66D04CX3CR1BII 46 GSIFSSNAMA 141 VINSVGITK 163 DARRGWDTRY 187 PMP66D02CX3CR1BII 47 GSIFSSNAMA 141 SIDSVGITK 175 DARRGDTRYW 187 PMP66D06CX3CR1BII 48 GSIFSSNAMA 141 LINSVGIKT 167 DGRRGWDTRY 189 PMP66G01 *CDRsequences were determined according to Antibody Engineering, vol 2 byKonetermann & Dübel (Eds.), Springer Verlag Heidelberg Berlin, 2010. Thesequence numbers in the table (SEQ) refer to the sequences in thesequence listing of the instant application.

TABLE 2 Family 9 SEQ SEQ SEQ Nanobody SEQ CDR1* CDR1 CDR2* CDR2 CDR3*CDR3 CX3CR1BII 49 GRTFSSYAMG 146 GISGSASRKY 176 SNSYPKVQFDY 191 PMP11H11CX3CR1BII 50 GRTFSSYAMG 146 GISGSASRKY 176 SNSYPKVQFDY 191 PMP12B6CX3CR1BII 51 GRTFSSYAMG 146 GISGSGSRKY 177 SNSYPKVQFDY 191 PMP12G9CX3CR1BII 52 GRTFSSYAMG 146 GISGSGSRKY 177 SNSYPKVQFDY 191 PMP15G11 *CDRsequences were determined according to Antibody Engineering, vol 2 byKonetermann & Dübel (Eds.), Springer Verlag Heidelberg Berlin, 2010. Thesequence numbers in the table (SEQ) refer to the sequences in thesequence listing of the instant application.

TABLE 3 Family 13 SEQ SEQ SEQ Nanobody SEQ CDR1* CDR1 CDR2* CDR2 CDR3*CDR3 CX3CR1BII 53 GTIFSNNAMG 147 SISSSGSTN 178 DARRGWNTAY 192 PMP18E6CX3CR1BII 54 GTIFSNTAMG 148 SISNSGSTN 179 DARRGWNSGY 193 PMP12C2CX3CR1BII 55 GIIFSNNAMG 149 SISNSGSTN 179 DARRGWNTAY 192 PMP18A10CX3CR1BII 56 GIIFSNNAMG 149 SIGSTYSTN 180 DARRGWNTAY 192 PMP18A2CX3CR1BII 57 RTIFRSNAMG 150 SISNSGSTN 179 DARRGWNTGY 194 PMP18A8CX3CR1BII 58 GIIFSNNAMG 149 SISSTYSTN 181 DARRGWNTAY 192 PMP18A9CX3CR1BII 59 GTIFRSNAMG 151 SISNSGSTN 179 DARRGWNSGY 193 PMP18B7CX3CR1BII 60 GTIFSNNAMG 147 SISSSGSTN 178 DARRGWNTAY 192 PMP18B9CX3CR1BII 61 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTAY 192 PMP18C6CX3CR1BII 62 GIIFSNNAMG 149 SISNSGSTN 179 DARRGWNTAY 192 PMP18C9CX3CR1BII 63 GIIFSNNAMG 149 SISNSGSTN 179 DARRGWNTAY 192 PMP18D1CX3CR1BII 64 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTAY 192 PMP18D10CX3CR1BII 65 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTAY 192 PMP18D12CX3CR1BII 66 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTAY 192 PMP18F1CX3CR1BII 67 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTAY 192 PMP18F5CX3CR1BII 68 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTAY 192 PMP18F6CX3CR1BII 69 GTIFRTNAMG 152 SISNSGSTN 179 DGRRGWNTGY 195 PMP18F9CX3CR1BII 70 RTIFRSNAMG 150 SISNSGSTN 179 DARRGWNTGY 194 PMP18G5CX3CR1BII 71 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTAY 192 PMP18H1CX3CR1BII 72 KTIFRSNAMG 153 SISNSGSTN 179 DARRGWNTGY 194 PMP18H10CX3CR1BII 73 GIIFSNNAMG 149 SISNSGSTN 179 DARRGWNTAY 192 PMP18H7CX3CR1BII 74 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTAY 192 PMP18H9CX3CR1BII 75 GIIFSNNAMG 149 SIGSTYSTN 180 DARRGWNTAY 192 PMP20B3CX3CR1BII 76 GTIFRSNAMG 151 SISNSGSTN 179 DARRGWNSGY 193 PMP20C12CX3CR1BII 77 GIIFSNNAMG 149 SISNSGSTN 179 DARRGWNTAY 192 PMP20C3CX3CR1BII 78 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTAY 192 PMP20C6CX3CR1BII 79 GTTFRSNAMG 154 SITNSGSTN 182 DARRGWNTGY 194 PMP20D8CX3CR1BII 80 RTIFRSNAMG 150 SITNSGSTN 182 DARRGWNTGY 194 PMP20E11CX3CR1BII 81 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTGY 194 PMP20E5CX3CR1BII 82 GTIFSNNAMG 147 SISSSGSTN 178 DARRGWNTAY 192 PMP20F3CX3CR1BII 83 ATIFRSNAMG 155 SISNSGSTN 179 DGRRGWNTGY 195 PMP20F4CX3CR1BII 84 ATIFRSNAMG 155 SISNSGSTN 179 DGRRGWNTGY 195 PMP20F5CX3CR1BII 85 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTAY 192 PMP21B6CX3CR1BII 86 GIIFSNNAMG 149 SISNSGSAN 183 DARRGWNTAY 192 PMP24Al2CX3CR1BII 87 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTAY 192 PMP24A6CX3CR1BII 88 GTIFRSNAMG 151 SISISGSTN 184 DARRGWNTGF 196 PMP24B9CX3CR1BII 89 GIIFSNNAMG 149 SISSTYSTN 181 DARRGWNTAY 192 PMP24D3CX3CR1BII 90 GLIFSNNAMG 156 SISSTYSTN 181 DARRGWNTAY 192 PMP24F7CX3CR1BII 91 ATIFRSNAMG 155 SISNSGSTN 179 DGRRGWNTGY 195 PMP28B4CX3CR1BII  92 GIIFSNNAMG 149 SIGSTYSTN 180 DARRGWNTAY 192 PMP28F1CX3CR1BII 93 GIIFSNNAMG 149 SISNSGSTN 179 DARRGWNTAY 192 PMP28F6CX3CR1BII 94 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTGY 194 PMP28F9CX3CR1BII 95 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTAY 192 PMP29A5CX3CR1BII 96 GTIFRSNAMG 151 SISNSGSTN 179 DARRGWNSGY 193 PMP29D5CX3CR1BII 97 KTIFRSNAMG 153 SISNSGSTN 179 DARRGWNTGY 194 PMP29E3CX3CR1BII 98 KTIFRSNAMG 153 SISNSGSTN 179 DARRGWNTGY 194 PMP29E7CX3CR1BII 99 GTIFRSNAMG 151 SITNSGSTN 182 DARRGWNTGY 194 PMP29G10CX3CR1BII 100 GIIFSNNAMG 149 SITNTGSTN 185 DARRGWNTAY 192 PMP29G7CX3CR1BII 101 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTAY 192 PMP29H1CX3CR1BII 102 RTIFRSNAMG 150 SISNSGSTN 179 DARRGWNTGY 194 PMP37A8CX3CR1BII 103 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTAY 192 PMP37B9CX3CR1BII 104 GSIFRSNAMG 157 SISNSGSTN 179 DARRGWNTGY 194 PMP37C12CX3CR1BII 105 RTIFSNNAMG 158 SISNSGSTN 179 DARRGWNTAY 192 PMP37C7CX3CR1BII 106 GTVFSNNAMG 159 SISSSGSTN 178 DARRGWNTAY 192 PMP37D9CX3CR1BII 107 KPIFRSNAMG 160 SISNSGSTN 179 DARRGWNTGY 194 PMP37E12CX3CR1BII 108 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTAY 192 PMP41B10CX3CR1BII 109 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTAY 192 PMP41B11CX3CR1BII 110 GIIFSNNAMG 149 SIGSTYSTN 180 DARRGWNTAY 192 PMP41B8CX3CR1BII 111 RTIFRSNAMG 150 SISNSGSTN 179 DARRGWNTGY 194 PMP41C10CX3CR1BII 112 GIIFSNNAMG 149 SIGSTYSTN 180 DARRGWNTAY 192 PMP41F9CX3CR1BII 113 GLTLDDYAMG 161 SISNSGSTN 179 DARRGWNTGY 194 PMP41H10CX3CR1BII 114 RTIFRSNAMG 150 SISNSGSTN 179 DARRGWNTGY 194 PMP46B5CX3CR1BII 115 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTGY 194 PMP46D3CX3CR1BII 116 GIIFSNNAMG 149 SISSTYSTN 181 DARRGWNTAY 192 PMP46H5CX3CR1BII 117 KTIFRSNAMG 153 SISNSGSTN 179 DARRGWNTGY 194 PMP48B8CX3CR1BII 118 RTIFRSNAMG 150 SISNSGSTN 179 DARRGWNTGY 194 PMP48D11CX3CR1BII 119 RTIFRSNAMG 150 SISNSGSTN 179 DARRGWNTGY 194 PMP48G8CX3CR1BII 120 GTIFSNNAMG 147 SISNSGSTN 179 DARRGWNTAY 192 PMP48H9 *CDRsequences were determined according to Antibody Engineering, vol 2 byKonetermann & Dübel (Eds.), Springer Verlag Heidelberg Berlin, 2010. Thesequence numbers in the table (SEQ) refer to the sequences in thesequence listing of the instant application.

TABLE 4 Optimized variants SEQ SEQ SEQ Nanobody SEQ CDR1 CDR1 CDR2 CDR2CDR3 CDR3 CX3CR1BII 1 GSIFSSNA 141 AINSVGV 162 DPRRGW 186 PMP66B02 MA TKDTRY CX3CR1BII 121 GSIFSSNA 141 AINSVGV 162 DPRRGW 186 043 MA TK DTRYCX3CR1BII 122 GSIFSSNA 141 AINSVGV 162 DPRRGW 186 045 MA TK DTRYCX3CR1BII 123 GSIFSSNA 141 AINSVGV 162 DPRRGW 186 047 MA TK DTRYCX3CR1BII 124 GSIFSSNA 141 AINSVGV 162 DPRRGW 186 048 MA TK DTRYCX3CR1BII 125 GSIFSSNA 141 AINSVGV 162 DPRRGW 186 049 MA TK DTRYCX3CR1BII 126 GSIFSSNA 141 AINSVGV 162 DPRRGW 186 050 MA TK DTRYCX3CR1BII 127 GSIFSSNA 141 AINSVGV 162 DPRRGW 186 061 MA TK DTRYCX3CR1BII 128 GSIFSSNA 141 AINSVGV 162 DPRRGW 186 056 MA TK DTRYCX3CR1BII 129 GSIFSSNA 141 AINSVGV 162 DPRRGW 186 057 MA TK DTRYCX3CR1BII 130 GSIFSSNA 141 AINSVGV 162 DPRRGW 186 060 MA TK DTRYCX3CR1BII 131 GSIFSSNA 141 AISSVGV 214 DPRRGW 186 065 MA TK DTRYCX3CR1BII 132 GSIFSSNA 141 AIQSVGV 215 DPRRGW 186 067 MA TK DTRYCX3CR1BII 133 GSIFSSNA 141 AIGSVGV 216 DPRRGW 186 068 MA TK DTRYCX3CR1BII 134 GSIFSSNA 141 AITSVGV 217 DPRRGW 186 074 MA TK DTRYCX3CR1BII 135 GSIFSSNA 141 AINTVGV 218 DPRRGW 186 118 MA TK DTRYCX3CR1BII 136 GSIFSSNA 141 AINGVGV 219 DPRRGW 186 129 MA TK DTRYCX3CR1BII 137 GSIFSSNA 141 AINPVGV 220 DPRRGW 186 158 MA TK DTRYCX3CR1BII 138 GSIFSSTA 213 AISSVGV 214 DPRRGW 186 306 MA TK DTRYCX3CR1BII 139 GSIFSSTA 213 AISTVGV 221 DPRRGW 186 307 MA TK DTRYCX3CR1BII 140 GSIFSSNA 141 AINSVGV 162 DPRRGW 186 308 MA TK DTRY *CDRsequences were determined according to Antibody Engineering, vol 2 byKonetermann & Dübel (Eds.), Springer Verlag Heidelberg Berlin, 2010. Thesequence numbers in the table (SEQ) refer to the sequences in thesequence listing of the instant application.

In a further aspect, the present invention provides polypeptides havingone or more VHH domains.

In one aspect, a VHH domain of the present invention comprises oressentially consists of the sequence set forth in:

-   -   a) the amino acid sequence of SEQ ID NO: 3; or    -   b) amino acid sequences that have at least 90% amino acid        identity with the amino acid sequences of SEQ ID NO: 3; or    -   c) amino acid sequences that have 11, 10, 9, 8, 7, 6, 5, 4, 3,        2, or 1 amino acid difference with the amino acid sequences of        SEQ ID NO: 3 or    -   d) an amino acid sequence of any one of SEQ ID NO: 1-48, or SEQ        ID NO: 121-140, or SEQ ID NO: 222-224.

In a further aspect, a VHH domain of the present invention comprises oressentially consists of the sequence set forth in:

-   -   a) the amino acid sequence of SEQ ID NO: 49; or    -   b) an amino acid sequence that has at least 95% amino acid        identity with the amino acid sequences of SEQ ID NO: 49; or    -   c) an amino acid sequence that has 5, 4, 3, 2, or 1 amino acid        difference with the amino acid sequences of SEQ ID NO: 49; or    -   d) an amino acid sequence of any one of SEQ ID NO: 49-52.

In a further aspect, a VHH domain of the present invention comprises oressentially consists of the sequence set forth in:

-   -   a) the amino acid sequence of SEQ ID NO: 67; or    -   b) an amino acid sequence that has at least 90% amino acid        identity with the amino acid sequences of SEQ ID NO: 67; or    -   c) an amino acid sequence that has 12, 11, 10, 9, 8, 7, 6, 5, 4,        3, 2, or 1 amino acid difference with the amino acid sequences        of SEQ ID NO: 67; or    -   d) an amino acid sequence of any one of SEQ ID NO: 53-120.

In a further aspect, a VHH domain of the present invention comprises oressentially consists of the amino acid sequence set forth in any one ofSEQ ID NO: 121-140, or SEQ ID NO: 222-224.

In a further aspect, a VHH domain of the present invention comprises oressentially consists of the amino acid sequence set forth in any one ofSEQ ID NO: 138-140.

In a further aspect, a VHH domain of the present invention comprises oressentially consists of the amino acid sequence set forth in any one ofSEQ ID NO: 222-224.

Representative VHH domains of the present invention are shown in Table 5and representative optimized VHH domains of the present invention areshown in Table 6 below:

TABLE 5 VHH domainsSEQ ID NO: 1-48 are VHH domains of family 101. SEQ ID NO: 49-52 are VHHdomains of family 9. SEQ ID NO: 53-120 are VHH domains of family 13.CX3CR1BII EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLVAAI SEQ ID NO:1 PMP66B02 NSVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAVI SEQ ID NO: 2PMP54A12 NSVGITKYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGWDTRYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAGI SEQ ID NO: 3 PMP54A3NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRG WDTRYWGQGTQVTVSSCX3CR1BII EVQLVESGRGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAGI SEQ ID NO:4 PMP54A4 NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAGI SEQ ID NO: 5 PMP54A5NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRG WDTRYWGQGTLVTVSSCX3CR1BII EVQLVESGGGSVQAGESLRLSCAASGTIFSSNAMAWYRQAPGKQRDLVAGI SEQ ID NO:6 PMP54A7 NSVDITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWNTRYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAGI SEQ ID NO: 7 PMP54B1NSVGITKYADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCTSDPRRG WDTRYWGQGTQVTVSSCX3CR1BII EVQLVESGGGSVQAGESLRLSCAASGTIFSSNAMAWYRQAPGKQRDLVAGI SEQ ID NO:8 PMP54B2 NSVDITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWNTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAAI SEQ ID NO: 9 PMP54B3NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRG WDTRYWGQGTQVTVSSCX3CR1BII EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAGI SEQ ID NO:10 PMP54B5 NSVGITKYADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPPGKQRDLVAL SEQ ID NO: 11PMP54D5 INSVGITKYADSVKGRFTISSDNAKNTVYLEMNSLKPEDTAVYYCTSDGRRGWDTRYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGSVQAGGSLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAGI SEQ ID NO: 12PMP54D8 NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIKVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAAI SEQ ID NO: 13PMP54F6 NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVALI SEQ ID NO: 14PMP54G3 NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGTIFSSNAMAWYRQAPGKQRDLVAGI SEQ ID NO: 15PMP54H1 NSVDITKYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWNTRYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAVI SEQ ID NO: 16PMP54H4 NSVGITKYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGWDTRYWGQGTLVTVSS CX3CR1BIIKVQLVESGGGSVQAGESLRLSCAASGTIFSSNAMAWYRQAPGKQRDLVAGI SEQ ID NO: 17PMP61F10 NSVDITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWNTRYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAFGKQRDLVALI SEQ ID NO: 18PMP61D1 NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSS CX3CR1BIIKVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAFGKQRDLVALI SEQ ID NO: 19PMP61D5 NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAGI SEQ ID NO: 20PMP61E2 NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDMAVYYCTSDPRRGWDTRYWGQGTQVTVSS CX3CR1BIIKVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQPPGKQRDLVAAI SEQ ID NO: 21PMP61F11 NSVGITKYADSVKGRFTIFRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSS CX3CR1BIIEVQLVKSGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVALI SEQ ID NO: 22PMP61G2 NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSS CX3CR1BIIKVQLVESGGGSMQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAAI SEQ ID NO: 23PMP61G3 NSVGITKYADSVKGRFTISRDNAKNTVLYQMMSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSS CX3CR1BIIKVQLVESGGGSVQAGGSLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAAI SEQ ID NO: 24PMP61G4 NSVGITKYADSVKGRFTISRDNAKNTVYLQMMSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGSVQAGASLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAVI SEQ ID NO: 25PMP61F4 NTVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESRGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAVI SEQ ID NO: 26PMP61A11 NSVGITKYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGWDTRYWGQGTQVTVSS CX3CR1BIIEVQLVESRGGSVQAGASLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAVI SEQ ID NO: 27PMP61B2 NTVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGWDTRYWGQGTQVTVSS CX3CR1BIIEVQLVKSGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQALGKQRDLVALI SEQ ID NO: 28PMP61C9 DSAGITKYADSVKGRFTISRDNAKNTVYLQMNRLKPEDTAVYYCASDARRGWNTKYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAAI SEQ ID NO: 29PMP65H02 NSVGITKYADSVKGRFTISRDNAKNTVHLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAGI SEQ ID NO: 30PMP65E11 NSVGIAKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAKAWYRQAPGKQRDLVAGI SEQ ID NO: 31PMP65E10 NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIKVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAGI SEQ ID NO: 32PMP65E05 NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVKSGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAVI SEQ ID NO: 33PMP65B11 NKVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAAI SEQ ID NO: 34PMP65B07 NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSRNAMAWYRQAPGKQRDLVASI SEQ ID NO: 35PMP65B09 NSVGITKYGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGGIFSRNAMAWYRQAPGKQRDLVASI SEQ ID NO: 36PMP65H01 NSVGITKYGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGWDTRYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGTIFSSNAMAWYRQAPGKQRDLVAGI SEQ ID NO: 37PMP65G07 NSVDITRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWNTRYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVALI SEQ ID NO: 38PMP66H08 NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGSVQAGGSLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAAI SEQ ID NO: 39PMP66H04 NSVGITKYADSVKGRFTISRDNAKNTVYLQMMSLKPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVALI SEQ ID NO: 40PMP66F02 NSVGITKYAGSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAAI SEQ ID NO: 41PMP66E11 NSVGTTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQALGKQRDLVALI SEQ ID NO: 42PMP66D10 NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSS CX3CR1BIIEVQLMESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAGI SEQ ID NO: 43PMP66D08 NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQALGKQRDLVALI SEQ ID NO: 44PMP66A04 NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIKVQLVESGGGSVQAGESLRLSCAASGTIFSSNAMAWYRQAPGKQRDLVAGI SEQ ID NO: 45PMP66D04 NSVDITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWNTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAVI SEQ ID NO: 46PMP66D02 NSVGITKYADSVKGRFTTSGDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGWDTRYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVASI SEQ ID NO: 47PMP66D06 DSVGITKYRDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGWDTRYWGQGTQVTVSS CX3CR1BIIEMQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVALI SEQ ID NO: 48PMP66G01 NSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDGRRGWDTRYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAMGWFRQAPGKERAFVAGI SEQ ID NO: 49PMP11H11 SGSASRKYYADSVKGRFTVSRDNARNTVYLQMNSLKPEDTAVYYCAASNSYPKVQFDYYGQGTQVTVSS CX3CR1BIIEVQLVQSGGGLVQAGGSLRLSCVASGRTFSSYAMGWFRQAPGRERAFVAGI SEQ ID NO: 50PMP12B6 SGSASRKYYADSVKGRFTVSRDNARNTVYLQMNSLKPEDTAVYYCAASNSYPKVQFDYYGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCVASGRTFSSYAMGWFRQAPGKEREFVAGI SEQ ID NO: 51PMP12G9 SGSGSRKYYADSVKGRFTISRDNARNTVYLQMNSLKPEDRAVYYCAASNSYPKVQFDYYGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAMGWFRQAPGKEREFVAGI SEQ ID NO: 52PMP15G11 SGSGSRKYYADSVKGRFTISRDNARNTVYLQMNSLKPEDRAVYYCAASNSYPKVQFDYYGQGTQVTVSS CX3CR1BIIKVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 53PMP18E6 SSSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTLDARRGWNTAYWGQGAQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGTIFSNTAMGWYRQAPGKKRDLVASI SEQ ID NO: 54PMP12C2 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNSGYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 55PMP18A10 SNSGSTNYADSAKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGVVQPGGSLRLSCVTSGIIFSNNAMGWYRQGPGKKRDLVASI SEQ ID NO: 56PMP18A2 GSTYSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGWNTAYWGQGTPVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 57PMP18A8 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGVVQPGGSLRLSCVTSGIIFSNNAMGWYRQGPGKKRDLVASI SEQ ID NO: 58PMP18A9 SSTYSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGWNTAYWGQGTPVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGTIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 59PMP18B7 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNSGYWGQGTQVTVSS CX3CR1BIIEVQLVESRGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 60PMP18B9 SSSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTLDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLMESGGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 61PMP18C6 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 62PMP18C9 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 63PMP18D1 SNSGSTNYADSVKGRFTVSRDNDKSTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLGLSCATSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 64PMP18D10 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCTTSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 65PMP18D12 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNNLKPEDTGVYYCTLDARRGWNTAYWGQGTQVTVSS CX3CR1BIIKVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 66PMP18F1 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 67PMP18F5 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVDSGGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 68PMP18F6 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGTIFRTNAMGWYRQAPGKKRDLVASI SEQ ID NO: 69PMP18F9 SNSGSTNYADSVKGRFTVSRDNDKNTAYLQMNSLKPEDTGVYYCTIDGRRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 70PMP18G5 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQALGKKRDLVASI SEQ ID NO: 71PMP18H1 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSKTIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 72PMP18H10 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESRGGLVQPGGSLRLSCATSGIIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 73PMP18H7 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVKSGGGLVQPGGSLRLSCTTSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 74PMP18H9 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNNLKPEDTGVYYCTLDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQAGGSLRLSCVTSGIIFSNNAMGWYRQGPGKKRDLVASI SEQ ID NO: 75PMP20B3 GSTYSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGWNTAYWGQGTPVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGTIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 76PMP20C12 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNSGYWGQGTRVTVSS CX3CR1BIIKVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 77PMP20C3 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQAGGSLRLSCATSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 78PMP20C6 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGRSLRLSCATSGTTFRSNAMGWYRQGPGKKRDLVASI SEQ ID NO: 79PMP20D8 TNSGSTNYADSVKGRFTVSRDNDKNTGYLQMSSLKPEDTGVYYCTLDARRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAMGWYRQGPGKKRDLVASI SEQ ID NO: 80PMP20E11 TNSGSTNYADSVKGRFTVSRDNDRNTGYLQMNSLKPEDTGVYYCTVDARRGWNTGYWGQGTQVTVSS CX3CR1BIIKVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQVPGKKRDLVASI SEQ ID NO: 81PMP20E5 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 82PMP20F3 SSSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTLDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSATIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 83PMP20F4 SNSGSTNYADSVKGRFTVSRDNDKNTAYLQMNSLKPEDTGVYYCTIDGRRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSATIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 84PMP20F5 SNSGSTNYADSVKGRSTVSRDNDKNTAYLQMNSLKPEDTGVYYCTIDGRRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 85PMP21B6 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDMGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 86PMP24A12 SNSGSANYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCTTSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 87PMP24A6 SNSGSTNYADSVKGRFTVSGDNDKNTGYLQMNNLKPEDTGVYYCTLDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGTIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 88PMP24B9 SISGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTGFWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCVTSGIIFSNNAMGWYRQGPGKKRDLVASI SEQ ID NO: 89PMP24D3 SSTYSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGWNTAYWGQGTPVTVSS CX3CR1BIIEVQLMESGGGMVQVGGSLRLSCTASGLIFSNNAMGWYRQGPGKKRDLVASI SEQ ID NO: 90PMP24F7 SSTYSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGWNTAYWGQGTPVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCAISATIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 91PMP28B4 SNSGSTNYADSVKGRFTVSRDNDKNTAYLQMNSLKPEDTGVYYCTIDGRRGWNTGYWGQGTQVTVSS CX3CR1BIIEMQLVESGGGVVQPGGSLRLSCVTSGIIFSNNAMGWYRQGPGKKRDLVASI SEQ ID NO: 92PMP28F1 GSTYSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGWNTAYWGQGTPVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 93PMP28F6 SNSGSTNHADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQVPGKKRDLVASI SEQ ID NO: 94PMP28F9 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESRGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 95PMP29A5 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIKVQLVESGGGLVQPGGSLRLSCATSGTIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 96PMP29D5 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNSGYWGQGTQVTVSS CX3CR1BIIEVQLVESEGGLVQPGGSLRLPCATSKTIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 97PMP29E3 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSKTIFRSNAMGWYRQAPGKKRGLVASI SEQ ID NO: 98PMP29E7 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLMESGGGLVQPGGSLRLSCATSGTIFRSNAMGWYRQGPGKKRDLVASI SEQ ID NO: 99PMP29G10 TNSGSTNYADSVKGRFTVSRDNDKNTGYLQMSSLKPEDTGVYYCTLDARRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGIIFSNNAMGWYRQGPGKKRDLVASI SEQ ID NO: 100PMP29G7 TNTGSTNYADSVKGRFTVSRDNDRNTVYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQAGGSLRLSCTTSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 101PMP29H1 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNNLKPEDTGVYYCTLDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 102PMP37A8 SNSGSTNYADSAKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 103PMP37B9 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQAGGSLRLSCVASGSIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 104PMP37C12 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSRTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 105PMP37C7 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGTVFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 106PMP37D9 SSSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTLDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSKPIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 107PMP37E12 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESEGGLVQPGGSLRLSCTTSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 108PMP41B10 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNNLKPEDTGVYYCTLDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 109PMP41B11 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSPKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS CX3CR1BIIEVQLVESEGGVVQPGGSLRLSCVTSGIIFSNNAMGWYRQGPGKKRDLVASI SEQ ID NO: 110PMP41B8 GSTYSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGWNTAYWGQGTPVTVSS CX3CR1BIIEMQLVESGGGLVQPGGSLRLSCATSRTIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 111PMP41C10 SNSGSTNYADSVKGRFTVSRDNDKSTGYLQMNSLKPEDTGVYYCTVDARRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGVVQPGESLRLSCVTSGIIFSNNAMGWYRQGPGKKRDLVASI SEQ ID NO: 112PMP41F9 GSTYSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGWNTAYWGQGTPVTVSS CX3CR1BIIKVQLVESGGGLVQPGDSLRLSCAASGLTLDDYAMGWYRQAPGKKRDLVASI SEQ ID NO: 113PMP41H10 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGWNTGYWGQGTQVTVSS CX3CR1BIIKVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 114PMP46B5 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQVPGKKRDLVASI SEQ ID NO: 115PMP46D3 SNSGSTNYADSVKGRFTVSRDNDKNTGYLRMNSLKPEDTGVYYCTVDARRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQAGGSLRLSCVTSGIIFSNNAMGWYRQGPGKKRDLVASI SEQ ID NO: 116PMP46H5 SSTYSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTIDARRGWNTAYWGQGTPVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSKTIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 117PMP48B8 SNSGSTNYTDSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTGYWGQGTQVTVSS CX3CR1BIIKVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 118PMP48D11 SNSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSRTIFRSNAMGWYRQAPGKKRDLVASI SEQ ID NO: 119PMP48G8 SNSGSTNYADSVKGRFAVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTGYWGQGTQVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQAPGKKRDLVASI SEQ ID NO: 120PMP48H9 SNSGSTNYADFVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTVDARRGWNTAYWGQGTQVTVSS

TABLE 6 Optimized VHH domains CX3CR1BIIEVQLVESGGGSVQPGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLV SEQ ID NO: 121 043AAINSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCT SDPRRGWDTRYWGQGTLVTVSSCX3CR1BII DVQLVESGGGSVQPGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLV SEQ ID NO:122 045 AAINSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGLVQPGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLV SEQ ID NO: 123 047AAINSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCT SDPRRGWDTRYWGQGTLVTVSSCX3CR1BII EVQLVESGGGSVQPGGSLRLSCAASGSIFSSNAMAWYRQAPGKRRDLV SEQ ID NO:124 048 AAINSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGSVQPGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLV SEQ ID NO: 125 049AAINSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCT SDPRRGWDTRYWGQGTLVTVSSCX3CR1BII EVQLVESGGGSVQPGESLRLSCAASGSIFSSNAMAWYRQAPGKRRELV SEQ ID NO:126 050 AAINSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAMAWYRQAPGKRRDLV SEQ ID NO:  127 061AAINSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCT SDPRRGWDTRYWGQGTLVTVSSCX3CR1BII EVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAMAWYRQAPGKQRDLV SEQ ID NO:128 056 AAINSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAMAWYRQAPGKRRELV SEQ ID NO: 129 057AAINSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCT SDPRRGWDTRYWGQGTLVTVSSCX3CR1BII EVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAMAWYRQAPGKQRELV SEQ ID NO:130 060 AAINSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLV SEQ ID NO: 131 065AAISSVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCT SDPRRGWDTRYWGQGTLVTVSSCX3CR1BII EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLV SEQ ID NO:132 067 AAIQSVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLV SEQ ID NO: 133 068AAIGSVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCT SDPRRGWDTRYWGQGTLVTVSSCX3CR1BII EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLV SEQ ID NO:134 074 AAITSVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLV SEQ ID NO: 135 118AAINTVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCT SDPRRGWDTRYWGQGTLVTVSSCX3CR1BII EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLV SEQ ID NO:136 129 AAINGVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLV SEQ ID NO: 137 158AAINPVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCT SDPRRGWDTRYWGQGTLVTVSSCX3CR1BII DVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRRDLV SEQ ID NO:138 306 AAISSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIDVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRRDLV SEQ ID NO: 139 307AAISTVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCT SDPRRGWDTRYWGQGTLVTVSSCX3CR1BII DVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAMAWYRQAPGKRRDLV SEQ ID NO:140 308 AAINSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRRDLV SEQ ID NO: 222 00306AAISSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCT (D1E)SDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRRDLV SEQ ID NO: 223 00307AAISTVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCT (D1E)SDPRRGWDTRYWGQGTLVTVSS CX3CR1BIIEVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAMAWYRQAPGKRRDLV SEQ ID NO: 224 00308AAINSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCT (D1E)SDPRRGWDTRYWGQGTLVTVSS

In a further aspect, a polypeptide according to the present invention,in particular an immunoglobulin single variable domain of the presentinvention, is humanized and/or optimized for stability, potency,manufacturability and/or similarity to human framework regions. Forexample, the polypeptide is humanized and/or sequence optimized in oneor more of the following positions (according to Kabat numbering): 1,11, 14, 16, 74, 83, 108. In one aspect, the polypeptide comprises one ormore of the following mutations: E1D, S11L, A14P, E16G, A74S, K83R,Q108L.

In one aspect, one or more framework regions of a polypeptide accordingto the present invention, in particular an immunoglobulin singlevariable domain of the present invention, are humanized and/or sequenceoptimized. In one aspect, a polypeptide according to the presentinvention, in particular an immunoglobulin single variable domain of thepresent invention, comprises framework regions (FR) for example as setforth below:

-   -   i) FR1 is selected from any one of SEQ ID NO's: 198-204;    -   ii) FR2 is selected from any one of SEQ ID NO's: 205-208;    -   iii) FR3 is selected form any one of SEQ ID NO's: 209-210;        and/or    -   iv) FR4 is selected from any one of SEQ ID NO's: 211-212.

Human immunoglobulin framework region sequences (FR) that can also beused as framework region sequences for the immunoglobulin singlevariable domains as described above are known in the art. Also known inthe art are methods for humanizing framework regions of immunoglobulinsingle variable domains derived from species other than humans.

In a further aspect, one or more CDR regions of a polypeptide accordingto the present invention, in particular an immunoglobulin singlevariable domain of the present invention, is humanized and/or sequenceoptimized. In one aspect, a polypeptide according to the presentinvention, in particular an immunoglobulin single variable domain of thepresent invention, is humanized and/or sequence optimized in one or moreof the following positions (according to Kabat numbering): 52, 53.

In a further aspect, a polypeptide according to the present invention,in particular an immunoglobulin single variable domain of the presentinvention, comprises one or more of the following mutations: N52S, S53T.

In a further aspect, a polypeptide according to the present invention,in particular an immunoglobulin single variable domain of the presentinvention, comprises a CDR2 selected from any one of SEQ ID NO's:214-221.

Representative humanized and/or optimized sequences of the presentinvention are shown in Table 4 and 6 hereinabove and in Table 7 hereinbelow.

Table 7: Sequence Optimized Variants

Table 7a shows the FR1-CDR1-FR2-CRD2 of the sequence optimized variants,table 7b shows FR3-CDR3-FR4-CDR4 of said variants. The sequence numbersin the tables (SEQ) refer to the sequences in the sequence listing ofthe instant application.

TABLE 7a Sequence optimized variants (FR1-CDR1-FR2-CDR2) Nano SEQ SEQSEQ SEQ body SEQ FR1 FR1 CDR1 CDR1 FR2 FR2 CDR2 CDR2 CX3CR1 1 EVQLVES198 GSIF 141 WYRQ 205 AINSV 162 BIIPMP6 GGGSVQA SSNA APGK GVTK 6B02GESLRLS MA RRDL CAAS VA CX3CR1 121 EVQLVES 199 GSIF 141 WYRQ 205 AINSV162 B11043 GGGSVQP SSNA APGK GVTK GESLRLS MA RRDL CAAS VA CX3CR1 122DVQLVES 200 GSIF 141 WYRQ 205 AINSV 162 B11045 GGGSVQP SSNA APGK GVTKGESLRLS MA RRDL CAAS VA CX3CR1 123 EVQLVES 201 GSIF 141 WYRQ 205 AINSV162 B11047 GGGLVQP SSNA APGK GVTK GESLRLS MA RRDL CAAS VA CX3CR1 124EVQLVES 202 GSIF 141 WYRQ 205 AINSV 162 B11048 GGGSVQP SSNA APGK GVTKGGSLRLS MA RRDL CAAS VA CX3CR1 125 EVQLVES 199 GSIF 141 WYRQ 206 AINSV162 B11049 GGGSVQP SSNA APGK GVTK GESLRLS MA QRDL CAAS VA CX3CR1 126EVQLVES 199 GSIF 141 WYRQ 207 AINSV 162 B11050 GGGSVQP SSNA APGK GVTKGESLRLS MA RREL CAAS VA CX3CR1 127 EVQLVES 203 GSIF 141 WYRQ 205 AINSV162 B11061 GGGLVQP SSNA APGK GVTK GGSLRLS MA RRDL CAAS VA CX3CR1 128EVQLVES 203 GSIF 141 WYRQ 206 AINSV 162 B11056 GGGLVQP SSNA APGK GVTKGGSLRLS MA QRDL CAAS VA CX3CR1 129 EVQLVES 203 GSIF 141 WYRQ 207 AINSV162 B11057 GGGLVQP SSNA APGK GVTK GGSLRLS MA RREL CAAS VA CX3CR1 130EVQLVES 203 GSIF 141 WYRQ 208 AINSV 162 B11060 GGGLVQP SSNA APGK GVTKGGSLRLS MA QREL CAAS VA CX3CR1 131 EVQLVES 198 GSIF 141 WYRQ 205 AISSV214 B11065 GGGSVQA SSNA APGK GVTK GESLRLS MA RRDL CAAS VA CX3CR1 132EVQLVES 198 GSIF 141 WYRQ 205 AIGSV 215 B11067 GGGSVQA SSNA APGK GVTKGESLRLS MA RRDL CAAS VA CX3CR1 133 EVQLVES 198 GSIF 141 WYRQ 205 AIGSV216 B11068 GGGSVQA SSNA APGK GVTK GESLRLS MA RRDL CAAS VA CX3CR1 134EVQLVES 198 GSIF 141 WYRQ 205 AITSV 217 B11074 GGGSVQA SSNA APGK GVTKGESLRLS M RRDL CAAS VA CX3CR1 135 EVQLVES 198 GSIF 141 WYRQ 205 AINTV218 B11118 GGGSVQA SSNA APGK GVTK GESLRLS MA RRDL CAAS VA CX3CR1 136EVQLVES 198 GSIF 141 WYRQ 205 AINGV 219 B11129 GGGSVQA SSNA APGK GVTKGESLRLS MA RRDL CAAS VA CX3CR1 137 EVQLVES 198 GSIF 141 WYRQ 205 AINPV220 B11158 GGGSVQA SSNA APGK GVTK GESLRLS MA RRDL CAAS VA CX3CR1 138DVQLVES 204 GSIF 213 WYRQ 205 AISSV 214 B11306 GGGLVQP SSTA APGK GVTKGGSLRLS MA RRDL CAAS VA CX3CR1 139 DVQLVES 204 GSIF 213 WYRQ 205 AISTV221 B11307 GGGLVQP SSTA APGK GVTK GGSLRLS MA RRDL CAAS VA CX3CR1 140DVQLVES 204 GSIF 141 WYRQ 205 AINSV 162 B11308 GGGLVQP SSNA APGK GVTKGGSLRLS MA RRDL CAAS VA

TABLE 7b Sequence optimized variants (FR3-CDR3-FR4) Nano SEQ SEQ SEQbody SEQ FR3 FR3 CDR3 CDR3 FR4 FR4 CX3CR1 1 YADSVKGRFTI 209 DPRRGW 186WGQGTQ 211 BIIPMP SRDNAKNTVYL DTRY VTVSS 66B02 QMNSLKPEDTA VYYCTS CX3CR1121 YADSVKGRFTI 210 DPRRGW 186 WGQGTL 212 BII043 SRDNSKNTVYL DTRY VTVSSQMNSLRPEDT AVYYCTS CX3CR1 122 YADSVKGRFTI 210 DPRRGW 186 WGQGTL 212BII045 SRDNSKNTVYL DTRY VTVSS QMNSLRPEDT AVYYCTS CX3CR1 123 YADSVKGRFTI210 DPRRGW 186 WGQGTL 212 BII047 SRDNSKNTVYL DTRY VTVSS QMNSLRPEDTAVYYCTS CX3CR1 124 YADSVKGRFTI 210 DPRRGW 186 WGQGTL 212 BII048SRDNSKNTVYL DTRY VTVSS QMNSLRPEDT AVYYCTS CX3CR1 125 YADSVKGRFTI 210DPRRGW 186 WGQGTL 212 BII049 SRDNSKNTVYL DTRY VTVSS QMNSLRPEDT AVYYCTSCX3CR1 126 YADSVKGRFTI 210 DPRRGW 186 WGQGTL 212 BII050 SRDNSKNTVYL DTRYVTVSS QMNSLRPEDT AVYYCTS CX3CR1 127 YADSVKGRFTI 210 DPRRGW 186 WGQGTL212 BII061 SRDNSKNTVYL DTRY VTVSS QMNSLRPEDT AVYYCTS CX3CR1 128YADSVKGRFTI 210 DPRRGW 186 WGQGTL 212 BII056 SRDNSKNTVYL DTRY VTVSSQMNSLRPEDT AVYYCTS CX3CR1 129 YADSVKGRFTI 210 DPRRGW 186 WGQGTL 212BII057 SRDNSKNTVYL DTRY VTVSS QMNSLRPEDT AVYYCTS CX3CR1 130 YADSVKGRFTI210 DPRRGW 186 WGQGTL 212 BII060 SRDNSKNTVYL DTRY VTVSS QMNSLRPEDTAVYYCTS CX3CR1 131 YADSVKGRFTI 209 DPRRGW 186 WGQGTL 212 BII065SRDNAKNTVYL DTRY VTVSS QMNSLKPEDTA VYYCTS CX3CR1 132 YADSVKGRFTI 209DPRRGW 186 WGQGTL 212 BII067 SRDNAKNTVYL DTRY VTVSS QMNSLKPEDTA VYYCTSCX3CR1 133 YADSVKGRFTI 209 DPRRGW 186 WGQGTL 212 BII068 SRDNAKNTVYL DTRYVTVSS QMNSLKPEDTA VYYCTS CX3CR1 134 YADSVKGRFTI 209 DPRRGW 186 WGQGTL212 BII074 SRDNAKNTVYL DTRY VTVSS QMNSLKPEDTA VYYCTS CX3CR1 135YADSVKGRFTI 209 DPRRGW 186 WGQGTL 212 BII118 SRDNAKNTVYL DTRY VTVSSQMNSLKPEDTA VYYCTS CX3CR1 136 YADSVKGRFTI 209 DPRRGW 186 WGQGTL 212BII129 SRDNAKNTVYL DTRY VTVSS QMNSLKPEDTA VYYCTS CX3CR1 137 YADSVKGRFTI209 DPRRGW 186 WGQGTL 212 BII158 SRDNAKNTVYL DTRY VTVSS QMNSLKPEDTAVYYCTS CX3CR1 138 YADSVKGRFTI 210 DPRRGW 186 WGQGTL 212 BII306SRDNSKNTVYL DTRY VTVSS QMNSLRPEDT AVYYCTS CX3CR1 139 YADSVKGRFTI 210DPRRGW 186 WGQGTL 212 BII307 SRDNSKNTVYL DTRY VTVSS QMNSLRPEDT AVYYCTSCX3CR1 140 YADSVKGRFTI 210 DPRRGW 186 WGQGTL 212 BII308 SRDNSKNTVYL DTRYVTVSS QMNSLRPEDT AVYYCTS

In one aspect of the present invention, a polypeptide of the inventioncan additionally contain modifications such as glycosyl residues,modified amino acid side chains, and the like.

It will be clear to the skilled person that for pharmaceutical uses inhumans, the polypeptides of the invention are preferably directedagainst human CX3CR1, whereas for veterinary purposes, the polypeptidesof the invention are preferably directed against CX3CR1 from the speciesto be treated.

It will also be clear to the skilled person that when used as atherapeutic agent in humans, the immunoglobulin single variable domainscomprised in the polypeptides according to the invention are preferablyhumanized immunoglobulin single variable domains.

According to the invention, an immunoglobulin single variable domain canbe a domain antibody, i.e. VL or VH antibody, and/or VHH domains asdescribed above, and/or any other sort of immunoglobulin single variabledomain, for example camelized VH, provided that these immunoglobulinsingle variable domains are anti-CX3CR1 immunoglobulin single variabledomains.

In one aspect of the invention, the immunoglobulin single variabledomain essentially consists of either a domain antibody sequence or aVHH domain sequence as described above. In particular, theimmunoglobulin single variable domain essentially consists of a VHHdomain sequences.

In a further aspect, a polypeptide of the present invention comprisestwo or more anti-CX3CR1 immunoglobulin single variable domains. In afurther aspect, a polypeptide of the present invention comprises twoanti-CX3CR1 immunoglobulin single variable domains, for exampleanti-CX3CR1 VHHs. In one aspect, the two anti-CX3CR1 immunoglobulinsingle variable domains in a polypeptide of the present invention havethe same amino acid sequence. In another aspect, the two anti-CX3CR1immunoglobulin single variable domains in a polypeptide of the presentinvention have different amino acid sequences.

According to another embodiment of the invention, the at least twoimmunoglobulin single variable domains present in a polypeptide of theinvention can be linked to each other directly (i.e. without use of alinker) or via a linker. The linker is preferably a linker peptide andwill, according to the invention, be selected so as to allow binding ofthe at least two immunoglobulin single variable domains to CX3CR1,either within one and the same CX3CR1 molecule, or within two differentmolecules.

Suitable linkers will inter alia depend on the epitopes and,specifically, the distance between the epitopes on CX3CR1 to which theimmunoglobulin single variable domains bind, and will be clear to theskilled person based on the disclosure herein, optionally after somelimited degree of routine experimentation.

Also, when the two or more anti-CX3CR1 immunoglobulin single variabledomains are domain antibodies or VHH domains, they may also be linked toeach other via a third domain antibody or VHH domain (in which the twoor more immunoglobulin single variable domains may be linked directly tothe third domain antibody or VHH domain or via suitable linkers). Such athird domain antibody or VHH domain may for example be a domain antibodyor VHH domain that provides for an increased half-life, as furtherdescribed herein. For example, the latter domain antibody or VHH domainmay be a domain antibody or VHH domain that is capable of binding to a(human) serum protein such as (human) serum albumin or (human)transferrin, as further described herein.

Alternatively, the two or more anti-CX3CR1 immunoglobulin singlevariable domains may be linked in series (either directly or via asuitable linker) and the third (single) domain antibody or VHH domain(which may provide for increased half-life, as decribed above) may beconnected directly or via a linker to one of these two or moreaforementioned immunoglobulin sequences.

Suitable linkers are described herein in connection with specificpolypeptides of the invention and may—for example and withoutlimitation—comprise an amino acid sequence, which amino acid sequencepreferably has a length of 5 or more amino acids, 7 or more amino acids,9 or more amino acids, 11 or more amino acids, 15 or more amino acids orat least 17 amino acids, such as about 20 to 40 amino acids. However,the upper limit is not critical but is chosen for reasons of convenienceregarding e.g. biopharmaceutical production of such polypeptides.

The linker sequence may be a naturally occurring sequence or anon-naturally occurring sequence. If used for therapeutical purposes,the linker is preferably non-immunogenic in the subject to which thepolypeptide of the invention is administered.

One useful group of linker sequences are linkers derived from the hingeregion of heavy chain antibodies as described in WO 96/34103 and WO94/04678.

Other examples are poly-alanine linker sequences such as Ala-Ala-Ala.

Further preferred examples of linker sequences are Gly/Ser linkers ofdifferent length such as (gly_(x)ser_(y))_(z) linkers, including(gly₄ser)₃ , (gly₄ser)₄, (gly₄ser), (gly₃ser), gly₃, and (gly₃ser₂)₃.

If the polypeptide of the invention is modified by the attachment of apolymer, for example a polyethylene glycol (PEG) moiety, the linkersequence preferably includes an amino acid residue, such as a cysteineor a lysine, allowing such modification, e.g. PEGylation, in the linkerregion.

Examples of Linkers are:

(5 GS linker, SEQ ID NO: 233) GGGGS (7GS linker, SEQ ID NO: 234) SGGSGGS(8GS linker, SEQ ID NO: 235) GGGGCGGGS (9GS linker, SEQ ID NO: 236)GGGGSGGGS (10GS linker, SEQ ID NO: 237) GGGGSGGGGS(15GS linker, SEQ ID NO: 238) GGGGSGGGGSGGGGS(18GS linker, SEQ ID NO: 239) GGGGSGGGGSGGGGGGGS(20GS linker, SEQ ID NO: 240) GGGGSGGGGSGGGGSGGGGS(25GS linker, SEQ ID NO: 241) GGGGSGGGGSGGGGSGGGGSGGGGS(30GS linker, SEQ ID NO: 242) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(35GS linker, SEQ ID NO: 243) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(G1 hinge linker, SEQ ID NO: 244) EPKSCDKTHTCPPCP(9GS-G1 hinge linker, SEQ ID NO: 245) GGGGSGGGSEPKSCDKTHTCPPCP(Llama upper long hinge region, SEQ ID NO: 246) EPKTPKPQPAAA(G3 hinge, SEQ ID NO: 247)ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEP KSCDTPPPCPRCP(Ala linker, SEQ ID NO: 248) AAA

Furthermore, the linker may also be a poly(ethylene glycol) moiety, asshown in e.g. WO04/081026.

Non-limiting examples of polypeptides comprising or consisting of two ormore anti-CX3CR1 immunoglobulin single variable domains are given inTable 8a.

TABLE 8a Bivalent anti-CX3CR1 polypeptides CX3CR1EVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAMG SEQ 267 BII007WFRQAPGKERAFVAGISGSASRKYYADSVKGRFTV IDSRDNARNTVYLQMNSLKPEDTAVYYCAASNSYPKV NO:QFDYYGQGTQVTVSSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSKVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQAPGKKRDLVASIS SSGSTNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTLDARRGWNTAYWGQGAQVTVSS CX3CR1KVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMG SEQ 268 BII009WYRQAPGKKRDLVASISSSGSTNYADSVKGRFTVS IDRDNDKNTGYLQMNSLKPEDTGVYYCTLDARRGWNT NO: AYWGQGAQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGS LRLSCVASGRTFSSYAMGWFRQAPGKERAFVAGISGSASRKYYADSVKGRFTVSRDNARNTVYLQMNSLK PEDTAVYYCAASNSYPKVQFDYYGQGTQVTVSSCX3CR1 EVQLVESGGGSVQAGGSLRLSCAASGSIFSSNAMA SEQ 269 BII012WYRQAPGKQRDLVAGINSVGITKYADSVKGRFTISR IDDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTR NO:YWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSKVQLVESGGGLVQPGGSLRLSCATSGTIFSNNAMGWYRQAPGKKRDLVASISSSGS TNYADSVKGRFTVSRDNDKNTGYLQMNSLKPEDTGVYYCTLDARRGWNTAYWGQGAQVTVSS CX3CR1 EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMASEQ 270 BII016 WYRQAPGKQRDLVAVINSVGITKYADSVKGRFTISG IDDNAKNTVYLQMNSLKPEDTAVYYCTSDARRGWDTR NO: YWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGESL RLSCAASGSIFSSNAMAWYRQAPGKQRDLVAVINSVGITKYADSVKGRFTISGDNAKNTVYLQMNSLKPEDT AVYYCTSDARRGWDTRYWGQGTQVTVSS CX3CR1EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMA SEQ 271 BII017WYRQAPPGKQRDLVALINSVGITKYADSVKGRFTIS IDSDNAKNTVYLEMNSLKPEDTAVYYCTSDGRRGWDTR NO:YWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPPGKQRDLVALINSVGITKYADSVKGRFTISSDNAKNTVYLEMNSLKPED TAVYYCTSDGRRGWDTRYWGQGTQVTVSSCX3CR1 EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMA SEQ 272 BII018WYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTISR IDDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTR NO: YWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGESL RLSCAASGSIFSSNAMAWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCTSDPRRGWDTRYWGQGTQVTVSS CX3CR1EMQLVESGGGSVQAGESLRLSCAASGSIFSSNAMA SEQ 273 BII019WYRQAPGKQRDLVALINSVGITKYADSVKGRFTISR IDDNAKNTVYLQMNSLKPEDTAVYYCTSDGRRGWDTR NO: YWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEMQLVESGGGSVQAGESL RLSCAASGSIFSSNAMAWYRQAPGKQRDLVALINSVGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCTSDGRRGWDTRYWGQGTQVTVSS CX3CR1EVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMA SEQ 274 BII020WYRQAPGKQRDLVAGINSVGITKYADSVKGRFTISR IDDNAKNTAYLQMNSLKPEDTAVYYCTSDPRRGWDTR NO:YWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAV YYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1EVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAMG SEQ 275 BII026WFRQAPGKERAFVAGISGSASRKYYADSVKGRFTV IDSRDNARNTVYLQMNSLKPEDTAVYYCAASNSYPKV NO:QFDYYGQGTQVTVSSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKPE DTAVYYCTSDPRRGWDTRYWGQGTQVTVSSCX3CR1 EVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAMG SEQ 276 BII027WFRQAPGKERAFVAGISGSASRKYYADSVKGRFTV IDSRDNARNTVYLQMNSLKPEDTAVYYCAASNSYPKV NO:QFDYYGQGTQVTVSSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKQRDLVAGINSVGITKYADSVKGRFTISRDNAKNTAYLQMNSLKPE DTAVYYCTSDPRRGWDTRYWGQGTLVTVSSCX3CR1 EVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAMG SEQ 282 BII006WFRQAPGKERAFVAGISGSASRKYYADSVKGRFTV IDSRDNARNTVYLQMNSLKPEDTAVYYCAASNSYPKV NO:QFDYYGQGTLVTVSSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCVASGRTFSSYAMGWFRQAPGKERAFVAGI SGSASRKYYADSVKGRFTVSRDNARNTVYLQMNSLKPEDTAVYYCAASNSYPKVQFDYYGQGTLVTVSS

In another embodiment, the at least two immunoglobulin single variabledomains of the polypeptide of the invention are linked to each other viaanother moiety (optionally via one or two linkers), such as anotherpolypeptide which, in a preferred but non-limiting embodiment, may be afurther immunoglobulin single variable domain as already describedabove. Such moiety may either be essentially inactive or may have abiological effect such as improving the desired properties of thepolypeptide or may confer one or more additional desired properties tothe polypeptide. For example, and without limitation, the moiety mayimprove the half-life of the protein or polypeptide, and/or may reduceits immunogenicity or improve any other desired property.

In one aspect, a polypeptide of the invention includes, especially whenused as a therapeutic agent, a moiety which extends the half-life of thepolypeptide of the invention in serum or other body fluids of a patient.The term “half-life” means the time taken for the serum concentration ofthe (modified) polypeptide to reduce by 50%, in vivo, for example due todegradation of the polypeptide and/or clearance and/or sequestration bynatural mechanisms.

According to a further embodiment of the invention, the twoimmunoglobulin single variable domains may be fused to a serum albuminmolecule, such as described e.g. in WO01/79271 and WO03/59934.

Alternatively, such half-life extending moiety can be covalently linkedor fused to said polypeptide and may be, without limitation, an Fcportion, an albumin moiety, a fragment of an albumin moiety, an albuminbinding moiety, such as an anti-albumin immunoglobulin single variabledomain, a transferrin binding moiety, such as an anti-transferrinimmunoglobulin single variable domain, a polyoxyalkylene molecule, suchas a polyethylene glycol molecule, an albumin binding peptide, orhydroxyethyl starch (HES) derivatives.

In another aspect, the polypeptide of the invention comprises a moietywhich binds to an antigen found in blood, such as serum albumin, serumimmunoglobulins, thyroxine-binding protein, fibrinogen or transferrin,thereby conferring an increased half-life in vivo to the resultingpolypeptide of the invention. According to one embodiment, such moietyis an albumin-binding immunoglobulin and, in particular, analbumin-binding immunoglobulin single variable domain such as analbumin-binding VHH domain.

In another embodiment, the polypeptide of the invention comprises amoiety which binds to serum albumin, wherein such moiety is an albuminbinding peptide, as described e.g. in international patent publicationsWO2008/068280 and WO2009/127691.

If intended for use in humans, such albumin-binding immunoglobulinsingle variable domain (also called anti-albumin immunoglobulin singlevariable domain) will preferably bind to human serum albumin and willpreferably be a humanized albumin-binding VHH domain.

Immunoglobulin single variable domains binding to human serum albuminare known in the art and are described in further detail in e.g.WO2006/122786. A specifically useful albumin binding VHH domain consistsof or contains the amino acid sequence as set forth in any one of SEQ IDNO: 230-232:

TABLE 8b ALB-1 AVQLVESGGGLVQPGNSLRLSCAASGFTFRSFG SEQ 230MSWVRQAPGKEPEWVSSISGSGSDTLYADSVK ID GRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTINO: GGSLSRSSQGTQVTVSS ALB-11 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFG SEQ 231(human- MSWVRQAPGKGLEWVSSISGSGSDTLYADSVK ID izedGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI NO: ALB-1) GGSLSRSSQGTLVTVSS ALB-2AVQLVESGGGLVQGGGSLRLACAASERIFDLNL SEQ 232MGWYRQGPGNERELVATCITVGDSTNYADSVK ID GRFTISMDYTKQTVYLHMNSLRPEDTGLYYCKINO: RRTWHSELWGQGTQVTVSS

According to one embodiment, a polypeptide of the invention may belinked to one or more antibody parts, fragments or domains that conferone or more effector functions to the polypeptide of the inventionand/or may confer the ability to bind to one or more Fc receptors. Forexample, for this purpose, and without being limited thereto, theantibody parts may be or may comprise CH2 and/or CH3 domains of anantibody, such as from a heavy chain antibody (as described hereabove)and more preferably from a conventional human 4-chain antibody;specifically, the polypeptide of the invention may be linked to an Fcregion, for example from human IgG, from human IgE or from another humanIg. For example, WO 94/04678 describes heavy chain antibodies comprisinga Camelid VHH domain or a humanized derivative thereof, in which theCamelidae CH2 and/or CH3 domain have been replaced by human CH2 and/orCH3 domains, so as to provide an immunoglobulin that consists of 2 heavychains each comprising a—optionally humanized—VHH domain and human CH2and CH3 domains (but no CH1 domain), which immunoglobulin has theeffector function provided by the CH2 and CH3 domains, can functionwithout the presence of any light chains, and has an increased half-lifeas compared to the corresponding VHH domains without such modification.

In one aspect, a polypeptide of the present invention comprises twoanti-CX3CR1 VHHs and a VHH capable of binding to serum albumin. In oneaspect, the VHHs are fused using linker peptides. Representativeexamples of such polypeptides of the present invention are shownhereinbelow.

In one aspect, a polypeptide of the present invention comprises a firstanti-CX3CR1 VHH fused to a first linker peptide, which is itself fusedto a VHH capable of binding to serum albumin, which is itself fused to asecond linker peptide, which is itself fused to a second anti-CX3CR1VHH. In one aspect, the first or the second linker peptide is a 9GSlinker, in one aspect, the first and the second linker peptide is a 9GSlinker. In one aspect, the VHH capable of binding to serum albumin iscapable of binding to human serum albumin. In one aspect, the VHHcapable of binding to serum albumin has the amino acid sequence setforth in SEQ ID NO: 231. In one aspect, the first and the secondanti-CX3CR1 VHH have the same amino acid sequence. In one aspect, thefirst or the second anti-CX3CR1 VHH has the CDR1, CDR2 and CDR3 setforth in:

-   -   SEQ ID NO's: 213, 214 and 186; or    -   SEQ ID NO's: 213, 221 and 186; or    -   SEQ ID NO's: 141, 162 and 186.

In one aspect, the first and the second anti-CX3CR1 VHH have the CDR1,CDR2 and CDR3 set forth in:

-   -   SEQ ID NO's: 213, 214 and 186; or    -   SEQ ID NO's: 213, 221 and 186; or    -   SEQ ID NO's: 141, 162 and 186.

In one aspect, the first or the second anti-CX3CR1 VHH has the aminoacid sequence set forth in any one of SEQ ID NO: 138 to 140 or SEQ IDNO: 222 to 224. In one aspect, the first and the second anti-CX3CR1 VHHhave the same amino acid sequence, wherein said amino acid sequence isthe sequence set forth in any one of SEQ ID NO: 138 to 140 or SEQ ID NO:222 to 224.

Non-limiting examples of polypeptides of the present invention are thepolypeptides of any one of SEQ ID NO: 225 to 227, 249 or 277 to 281.

TABLE 9 CX3CR1 DVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA SEQ 225 BII00312MAWYRQAPGKRRDLVAAISSVGVTKYADSVKGR ID FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRNO: RGWDTRYWGQGTLVTVSSGGGGSGGGSEVQL VESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRRDLVAAISSVGVTKYADSVKGRFTISRDNSKNTVYL QMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1 DVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA SEQ 226 BII00313MAWYRQAPGKRRDLVAAISTVGVTKYADSVKGR ID FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRNO: RGWDTRYWGQGTLVTVSSGGGGSGGGSEVQL VESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRRDLVAAISTVGVTKYADSVKGRFTISRDNSKNTVYL QMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1 DVQLVESGGGLVQPGGSLRLSCAASGSIFSSNA SEQ 227 BII00314MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR ID FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRNO: RGWDTRYWGQGTLVTVSSGGGGSGGGSEVQL VESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGSIFSSNAMAWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTISRDNSKNTV YLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1 EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA SEQ 277 BII032MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR ID FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRNO: RGWDTRYWGQGTQVTVSSGGGGSGGGSEVQL VESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTISR DNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSSGGGGSGGGSEVQLVES GGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRD NAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS CX3CR1 EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA SEQ 278 BII034MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR ID FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRNO: RGWDTRYWGQGTQVTVSSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQ APGKRRDLVAAINSVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTR YWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPG KGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTL VTVSS CX3CR1EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA SEQ 249 BII036MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR ID FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRNO: RGWDTRYWGQGTLVTVSSGGGGSGGGSEVQL VESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGSV QAGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTISRDNAKNTVY LQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTLVTVSS CX3CR1 EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA SEQ 279 BII040MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR ID FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRNO: RGWDTRYWGQGTQVTVSSGGGGSGGGSEVQL VESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTISR DNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTRYWGQGTQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGK GLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLV TVSS CX3CR1EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA SEQ 280 BII041MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR ID FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRNO: RGWDTRYWGQGTQVTVSSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGESLRLSCAASGSIFSSNAMAWYRQ APGKRRDLVAAINSVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRRGWDTR YWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQP GNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLY LQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVS SCX3CR1 EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA SEQ 281 BII042MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR ID FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRNO: RGWDTRYWGQGTQVTVSSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQ APGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQ GTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGESLRL SCAASGSIFSSNAMAWYRQAPGKRRDLVAAINSVGVTKYADSVKGRFTISRDNAKNTVYLQMNSLKP EDTAVYYCTSDPRRGWDTRYWGQGTQVTVSS

In another aspect, a polypeptide of the present invention comprises ananti-CX3CR1 VHH and a Fc domain. In one aspect, a polypeptide of thepresent invention comprises an anti-CX3CR1 VHH fused to a linkerpeptide, which is itself fused to a Fc domain. In one aspect, the linkerpeptide is a 15GS linker. In one aspect, the Fc domain has the aminoacid sequence set forth in SEQ ID NO: 250 or 252. In one aspect, the VHHhas the CDR1, CDR2 and CDR3 set forth in:

-   -   SEQ ID NO's: 213, 214 and 186; or    -   SEQ ID NO's: 213, 221 and 186; or    -   SEQ ID NO's: 141, 162 and 186.

In one aspect, the VHH has the amino acid sequence set forth in any oneof SEQ ID NO: 138 to 140 or SEQ ID NO: 222 to 224. In one aspect thepolypeptide is in the form of a dimer, for example wherein the dimer isformed by one or more disulfide bridge.

Non-limiting examples of polypeptides of the present invention are thepolypeptides of SEQ ID NO: 251, 253 or 254.

TABLE 10 Mouse Fc PPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPI SEQ 250 domainVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTH ID REDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKNO: DLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTK KQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCS VVHEGLHNHHTTKSFSRTPGK 66B02-EVQLVESGGGSVQAGESLRLSCAASGSIFSSNA SEQ 251 mFcMAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR ID FTISRDNAKNTVYLQMNSLKPEDTAVYYCTSDPRNO: RGWDTRYWGQGTLVTVSSGGGGSGGGGSGG GGSPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQ THREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEE MTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSY SCSVVHEGLHNHHTTKSFSRTPGK Human FcCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV SEQ 252 DomainTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR ID EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNNO: KALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 306D-hFcDVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA SEQ 253MAWYRQAPGKRRDLVAAISSVGVTKYADSVKGR ID FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRNO: RGWDTRYWGQGTLVTVSSGGGGSGGGGSGG GGSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 307D-hFcDVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA SEQ 254MAWYRQAPGKRRDLVAAISTVGVTKYADSVKGR ID FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRNO: RGWDTRYWGQGTLVTVSSGGGGSGGGGSGG GGSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK

A polypeptide of the invention may be modified to improve itsproperties. In one aspect, a polypeptide of the present invention may bemodified to increase its stability upon storage. In one aspect, apolypeptide of the present invention may be modified to facilitate itsexpression in a particular host system. For example, the first codon ofa polypeptide of the present invention may be modified. In one aspect, apolypeptide of the present invention begins with a glutamic acid (glu)as its first amino acid. In another aspect, a polypeptide of the presentinvention begins with an aspartic acid (asp) as its first amino acid,for example to reduce pyroglutamate formation at the N-terminus duringstorage and hence increase product stability. In another aspect, apolypeptide of the present invention begins with an alanine (ala) or avaline (val) as its first amino acid, for example to facilitate theexpression of the polypeptide in a prokaryotic expression system, suchas Escherichia coli. Such modification of a polypeptide according to thepresent invention are made using techniques known in the art.

Representative examples of polypeptides according to the presentinvention with a modified first codon are set forth in any one of SEQ IDNO: 257-262 and 263-266 and are shown in Tables 11 and 12 below:

TABLE 11 CX3CR1 AVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA SEQ 257 BII00312MAWYRQAPGKRRDLVAAISSVGVTKYADSVKGR ID (D1A)FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPR NO: RGWDTRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR SSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRR DLVAAISSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQG TLVTVSS CX3CR1AVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA SEQ 258 BII00313MAWYRQAPGKRRDLVAAISTVGVTKYADSVKGR ID (D1A)FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPR NO: RGWDTRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR SSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRR DLVAAISTVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQG TLVTVSS CX3CR1AVQLVESGGGLVQPGGSLRLSCAASGSIFSSNA SEQ 259 BII00314MAWYRQAPGKRRDLVAAINSVGVTKYADSVKGR ID (D1A)FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPR NO: RGWDTRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR SSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAMAWYRQAPGKR RDLVAAINSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQ GTLVTVSS CX3CR1VQLVESGGGLVQPGGSLRLSCAASGSIFSSTAM SEQ 260 BII00312AWYRQAPGKRRDLVAAISSVGVTKYADSVKGRF ID (D1V)TISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRR NO: GWDTRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWV RQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRRD LVAAISSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQG TLVTVSS CX3CR1VQLVESGGGLVQPGGSLRLSCAASGSIFSSTAM SEQ 261 BII00313AWYRQAPGKRRDLVAAISTVGVTKYADSVKGRF ID (D1V)TISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRR NO: GWDTRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWV RQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAMAWYRQAPGKRRD LVAAISTVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQGTL VTVSS CX3CR1VQLVESGGGLVQPGGSLRLSCAASGSIFSSNAM SEQ 262 BII00314AWYRQAPGKRRDLVAAINSVGVTKYADSVKGRF ID (D1V)TISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRR NO: GWDTRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWV RQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFSSNAMAWYRQAPGKRRD LVAAINSVGVTKYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRGWDTRYWGQG TLVTVSS

TABLE 12 306D-hFc AVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA SEQ 263 (D1A)MAWYRQAPGKRRDLVAAISSVGVTKYADSVKGR ID FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRNO: RGWDTRYWGQGTLVTVSSGGGGSGGGGSGG GGSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 307D-hFcAVQLVESGGGLVQPGGSLRLSCAASGSIFSSTA SEQ 264 (D1A)MAWYRQAPGKRRDLVAAISTVGVTKYADSVKGR ID FTISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRNO: RGWDTRYWGQGTLVTVSSGGGGSGGGGSGG GGSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 306D-hFcVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAM SEQ 265 (D1V)AWYRQAPGKRRDLVAAISSVGVTKYADSVKGRF ID TISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRNO: GWDTRYWGQGTLVTVSSGGGGSGGGGSGGG GSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 307D-hFcVQLVESGGGLVQPGGSLRLSCAASGSIFSSTAM SEQ 266 (D1V)AWYRQAPGKRRDLVAAISTVGVTKYADSVKGRF ID TISRDNSKNTVYLQMNSLRPEDTAVYYCTSDPRRNO: GWDTRYWGQGTLVTVSSGGGGSGGGGSGGG GSCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK

In one further aspect, a polypeptide of the present invention ischaracterized by one or more of the following properties:

-   -   Bind with high affinity to human CX3CR1;    -   Inhibit binding of soluble fractalkine to human CX3CR1;    -   Inhibit fractalkine induced chemotaxis;    -   Inhibit fractalkine induced human CX3CR1 receptor        internalization;    -   Cross-react with cyno CX3CR1 within 10-fold of E/IC₅₀ for human        CX3CR1 for binding and functional inhibition.

Accordingly, in one aspect, a polypeptide of the present invention hasan affinity to human CX3CR1 at an IC50 less than or equal to 10 nM, orless than or equal to 5 nM, or less than or equal to 2.5 nM or less thanor equal to 1 nM, as determined by competition FACS.

In a further aspect, a polypeptide of the present invention has anaffinity to human CX3CR1 at an EC50 of less than or equal to 10 nM, orless than or equal to 5 nM, or less than or equal to 2.5 nM or less thanor equal to 1 nM, as determined by cell binding FACS.

In a further aspect, a polypeptide of the present invention blocks thebinding of human CX3CR1 to human fractalkine at or above 50%, or at orabove 60%, or at or above 70%, or at or above 80%, or at or above 90%,or at or above 95% as determined by competition FACS with humanfractalkine.

In a further aspect, a polypeptide of the present invention blocks thebinding of human fractalkine to human CX3CR1 at an IC50 of less than orequal to 300 nM, or less than or equal to 100 nM, or less than or equalto 20 nM, or less than or equal to 10 nM, less than or equal to 5 nM,less than or equal to 2.5 nM or less than or equal to 1 nM as determinedby competition FACS with human fractalkine.

In a further aspect, a polypeptide of the present invention inhibitsfractalkine induced chemotaxis mediated by human CX3CR1 at or above 10%,or at or above 30%, or at or above 40%, or at or above 50%, or at orabove 60%, or at or above 70%, or at or above 80%, or at or above 90%.

In a further aspect, a polypeptide of the present invention inhibitsfractalkine induced chemotaxis mediated by human CX3CR1 at an 10₅₀ ofless than or equal to 500 nM, or of less than or equal to 100 nM, orless than or equal to 75 nM, or less than or equal to 50 nM, or lessthan or equal to 10 nM or less than or equal to 5 nM.

In a further aspect, a polypeptide of the present invention inhibitsfractalkine induced human CX3CR1 receptor internalization at an IC₅₀ ofless than or equal to 10 nM, or less than or equal to 5 nM or or lessthan or equal to 1 nM.

According to still another embodiment, a half-life extendingmodification of a polypeptide of the invention (such modification alsoreducing immunogenicity of the polypeptide) comprises attachment of asuitable pharmacologically acceptable polymer, such as straight orbranched chain poly(ethylene glycol) (PEG) or derivatives thereof (suchas methoxypoly(ethylene glycol) or mPEG). Generally, any suitable formof PEGylation can be used, such as the PEGylation used in the art forantibodies and antibody fragments (including but not limited to domainantibodies and scFv's); reference is made, for example, to: Chapman,Nat. Biotechnol., 54, 531-545 (2002); Veronese and Harris, Adv. DrugDeliv. Rev. 54, 453-456 (2003); Harris and Chess, Nat. Rev. Drug.Discov. 2 (2003); WO 04/060965; and US6,875,841.

Various reagents for PEGylation of polypeptides are also commerciallyavailable, for example from Nektar Therapeutics, USA, or NOFCorporation, Japan, such as the Sunbright® EA Series, SH Series, MASeries, CA Series, and ME Series, such as Sunbright® ME-100MA,Sunbright® ME-200MA, and Sunbright® ME-400MA.

Preferably, site-directed PEGylation is used, in particular via acysteine-residue (see for example Yang et al., Protein Engineering 16,761-770 (2003)). For example, for this purpose, PEG may be attached to acysteine residue that naturally occurs in a polypeptide of theinvention, a polypeptide of the invention may be modified so as tosuitably introduce one or more cysteine residues for attachment of PEG,or an amino acid sequence comprising one or more cysteine residues forattachment of PEG may be fused to the N- and/or C-terminus and/or PEGmay be attached to a linker region that bridges two or more functionaldomains of a polypeptide of the invention, all using techniques ofprotein engineering known per se to the skilled person.

Preferably, for the polypeptides of the invention, a PEG is used with amolecular weight of more than 5 kDa, such as more than 10 kDa and lessthan 200 kDa, such as less than 100 kDa; for example in the range of 20kDa to 80 kDa.

With regard to PEGylation, it should be noted that generally, theinvention also encompasses any polypeptide of the invention that hasbeen PEGylated at one or more amino acid positions, preferably in such away that said PEGylation either (1) increases the half-life in vivo; (2)reduces immunogenicity; (3) provides one or more further beneficialproperties known per se for PEGylation; (4) does not essentially affectthe affinity of the polypeptide for CX3CR1 (e.g. does not reduce saidaffinity by more than 50%, and more preferably not by more than 10%, asdetermined by a suitable assay, such as those described in the Examplesbelow); and/or (4) does not affect any of the other desired propertiesof the polypeptides of the invention. Suitable PEG-groups and methodsfor attaching them, either specifically or non-specifically, will beclear to the skilled person.

According to a specifically preferred embodiment of the invention, aPEGylated polypeptide of the invention includes one PEG moiety of linearPEG having a molecular weight of 40 kDa or 60 kDa, wherein the PEGmoiety is attached to the polypeptide in a linker region and,specifially, at a Cys residue, for example at position 5 of a GS8-linkerpeptide as shown in SEQ ID NO:235.

Preferred examples of PEGylated polypeptides of the invention arePEGylated preferably with one of the PEG reagents as mentioned above,such as “Sunbright® ME-400MA” as shown in the following chemicalformula:

which has an average molecular weight of 40 kDa.Therapeutic Uses

In one aspect, the present invention provides a polypeptide of thepresent invention or a pharmaceutical composition comprising saidpolypeptide for use as a medicament.

In one aspect, the present invention provides the use of a polypeptideof the present invention or a pharmaceutical composition comprising saidpolypeptide for the treatment or prophylaxis of cardio- andcerebrovascular atherosclerotic disorders, peripheral artery disease,restenosis, diabetic nephropathy, glomerulonephritis, human crescenticglomerulonephritis, IgA nephropathy, membranous nephropathy, lupusnephritis, vasculitis including Henoch-Schonlein purpura and Wegener'sgranulomatosis, rheumatoid arthritis, osteoarthritis, allograftrejection, systemic sclerosis, neurodegenerative disorders anddemyelinating disease, multiple sclerosis (MS), Alzheimer's disease,pulmonary diseases such as COPD, asthma, neuropathic pain, inflammatorypain, or cancer.

In another aspect, the present invention provides the use of apolypeptide of the present invention or a pharmaceutical compositioncomprising said polypeptide for the treatment or prophylaxis ofatherosclerosis.

In another aspect, the present invention provides the use of apolypeptide of the present invention or a pharmaceutical compositioncomprising said polypeptide for the treatment or prophylaxis ofatherosclerosis by preventing and/or reducing the formation of newatherosclerotic lesions or plaques and/or by preventing or slowingprogression of existing lesions and plaques.

In another aspect, the present invention provides the use of apolypeptide of the present invention or a pharmaceutical compositioncomprising said polypeptide for the treatment or prophylaxis ofatherosclerosis by changing the composition of the plaques to reduce therisk of plaque rupture and atherothrombotic events.

In one aspect, the present invention also provides a method of treating,or reducing the risk of, cardio- and cerebrovascular atheroscleroticdisorders, peripheral artery disease, restenosis, diabetic nephropathy,glomerulonephritis, human crescentic glomerulonephritis, IgAnephropathy, membranous nephropathy, lupus nephritis, vasculitisincluding Henoch-Schonlein purpura and Wegener's granulomatosis,rheumatoid arthritis, osteoarthritis, allograft rejection, systemicsclerosis, neurodegenerative disorders and demyelinating disease,multiple sclerosis (MS), Alzheimer's disease, pulmonary diseases such asCOPD, asthma, neuropathic pain, inflammatory pain, or cancer, in aperson suffering from or at risk of, said disease or condition, whereinthe method comprises administering to the person a therapeuticallyeffective amount of a polypeptide according to the present invention ora pharmaceutical composition comprising said polypeptide.

In one aspect, the present invention also provides a method of treating,or reducing the risk of atherosclerosis in a person suffering from or atrisk of, said disease or condition, wherein the method comprisesadministering to the person a therapeutically effective amount ofpolypeptide of the present invention or a pharmaceutical compositioncomprising said polypeptide.

In one aspect, the present invention also provides a method of treating,or reducing the risk of atherosclerosis by preventing and/or reducingthe formation of new atherosclerotic lesions or plaques and/or bypreventing or slowing progression of existing lesions and plaques in aperson suffering from or at risk of, said disease or condition, whereinthe method comprises administering to the person a therapeuticallyeffective amount of polypeptide of the present invention or apharmaceutical composition comprising said polypeptide.

In one aspect, the present invention also provides a method of treating,or reducing the risk of atherosclerosis by changing the composition ofthe plaques so as to reduce the risk of plaque rupture andatherothrombotic events in a person suffering from or at risk of, saiddisease or condition, wherein the method comprises administering to theperson a therapeutically effective amount of a polypeptide of thepresent invention or a pharmaceutical composition comprising saidpolypeptide.

In one aspect, a polypeptide of the present invention is indicated foruse in the treatment or prophylaxis of a disease or disorder that isassociated with CX3CR1.

In one aspect, a polypeptide of the present invention is indicated foruse in the treatment or prophylaxis of diseases or conditions in whichmodulation of activity at the CX3CR1 receptor is desirable. In oneaspect, the present invention also provides a method of treating, orreducing the risk of, diseases or conditions in which antagonism of theCX3CR1 receptor is beneficial which comprises administering to a personsuffering from or at risk of, said disease or condition, a polypeptideof the present invention.

Prophylaxis is expected to be particularly relevant to the treatment ofpersons who have suffered a previous episode of, or are otherwiseconsidered to be at increased risk of, the disease or condition inquestion. Persons at risk of developing a particular disease orcondition generally include those having a family history of the diseaseor condition, or those who have been identified by genetic testing orscreening to be particularly susceptible to developing the disease orcondition.

In the context of the present invention, the term “prevention, treatmentand/or alleviation” not only comprises preventing and/or treating and/oralleviating the disease, but also generally comprises preventing theonset of the disease, slowing or reversing the progress of disease,preventing or slowing the onset of one or more symptoms associated withthe disease, reducing and/or alleviating one or more symptoms associatedwith the disease, reducing the severity and/or the duration of thedisease and/or of any symptoms associated therewith and/or preventing afurther increase in the severity of the disease and/or of any symptomsassociated therewith, preventing, reducing or reversing anyphysiological damage caused by the disease, and generally anypharmacological action that is beneficial to the patient being treated.

The subject to be treated will be a mammal, and more in particular ahuman being. As will be clear to the skilled person, the subject to betreated will in particular be a person suffering from, or at risk from,the diseases, disorders or conditions mentioned herein.

It will also be clear to the skilled person that the above methods oftreatment of a disease include the preparation of a medicament for thetreatment of said disease. Furthermore, it is clear that thepolypeptides of the invention can be used as an active ingredient in amedicament or pharmaceutical composition intended for the treatment ofthe above diseases. Thus, the invention also relates to the use of apolypeptide of the invention in the preparation of a pharmaceuticalcomposition for the prevention, treatment and/or alleviation of any ofthe diseases, disorders or conditions mentioned hereinabove. Theinvention further relates to a polypeptide of the invention fortherapeutic or prophylactic use and, specifically, for the prevention,treatment and/or alleviation of any of the diseases, disorders orconditions mentioned hereinabove. The invention further relates to apharmaceutical composition for the prevention, treatment and/oralleviation of the diseases, disorders or conditions mentionedhereinabove, wherein such composition comprises at least one polypeptideof the invention.

The polypeptides of the invention and/or the compositions comprising thesame can be administered to a patient in need thereof in any suitablemanner, depending on the specific pharmaceutical formulation orcomposition to be used.

Thus, the polypeptides of the invention and/or the compositionscomprising the same can for example be administered intravenously,subcutaneously, intramuscularly, intraperitoneally, transdermally,orally, sublingually (e.g. in the form of a sublingual tablet, spray ordrop placed under the tongue and adsorbed through the mucus membranesinto the capillary network under the tongue), (intra-)nasally (e.g. inthe form of a nasal spray and/or as an aerosol), topically, by means ofa suppository, by inhalation, intravitreally (esp. for the treatment ofdry AMD or glaucoma), or any other suitable manner in an effectiveamount or dose.

The polypeptides of the invention and/or the compositions comprising thesame are administered according to a regimen of treatment that issuitable for preventing, treating and/or alleviating the disease,disorder or condition to be prevented, treated or alleviated. Theclinician will generally be able to determine a suitable treatmentregimen, depending on factors such as the disease, disorder or conditionto be prevented, treated or alleviated, the severity of the disease, theseverity of the symptoms thereof, the specific polypeptide of theinvention to be used, the specific route of administration andpharmaceutical formulation or composition to be used, the age, gender,weight, diet, general condition of the patient, and similar factors wellknown to the clinician. Generally, the treatment regimen will comprisethe administration of one or more polypeptides of the invention, or ofone or more compositions comprising the same, in therapeutically and/orprohylactically effective amounts or doses.

Generally, for the prevention, treatment and/or alleviation of thediseases, disorders and conditions mentioned herein and depending on thespecific disease, disorder or condition to be treated, the potency ofthe specific polypeptide of the invention to be used, the specific routeof administration and the specific pharmaceutical formulation orcomposition used, the polypeptides of the invention will generally beadministered in an amount between 0.005 and 20.0 mg per kilogram of bodyweight and dose, preferably between 0.05 and 10.0 mg/kg/dose, and morepreferably between 0.5 and 10 mg/kg/dose, either continuously (e.g. byinfusion) or as single doses (such as e.g. daily, weekly, or monthlydoses; cf. below), but can significantly vary, especially, depending onthe before-mentioned parameters.

For prophylactic applications, compositions containing the polypeptidesof the invention may also be administered in similar or slightly lowerdosages. The dosage can also be adjusted by the individual physician inthe event of any complication.

Depending on the specific polypeptide of the invention and its specificpharmacokinetic and other properties, it may be administered daily,every second, third, fourth, fifth or sixth day, weekly, monthly, andthe like. An administration regimen could include long-term, weeklytreatment. By “long-term” is meant at least two weeks and preferablymonths, or years of duration.

The efficacy of the polypeptides of the invention, and of compositionscomprising the same, can be tested using any suitable in vitro assay,cell-based assay, in vivo assay and/or animal model known per se, or anycombination thereof, depending on the specific disease involved.Suitable assays and animal models will be clear to the skilled person,and for example include the assays and animal models used in theExamples below.

For pharmaceutical use, the polypeptides of the invention may beformulated as a pharmaceutical preparation comprising (i) at least onepolypeptide of the invention and (ii) at least one pharmaceuticallyacceptable carrier, diluent, excipient, adjuvant, and/or stabilizer, and(iii) optionally one or more further pharmaceutically activepolypeptides and/or compounds. By “pharmaceutically acceptable” is meantthat the respective material does not show any biological or otherwiseundesirable effects when administered to an individual and does notinteract in a deleterious manner with any of the other components of thepharmaceutical composition (such as e.g. the pharmaceutically activeingredient) in which it is contained. Specific examples can be found instandard handbooks, such as e.g. Remington's Pharmaceutical Sciences,18^(th) Ed., Mack Publishing Company, USA (1990). For example, thepolypeptides of the invention may be formulated and administered in anymanner known per se for conventional antibodies and antibody fragmentsand other pharmaceutically active proteins. Thus, according to a furtherembodiment, the invention relates to a pharmaceutical composition orpreparation that contains at least one polypeptide of the invention andat least one pharmaceutically acceptable carrier, diluent, excipient,adjuvant and/or stabilizer, and optionally one or more furtherpharmaceutically active substances.

By means of non-limiting examples, such a formulation may be in a formsuitable for oral administration, for parenteral administration (such asby intravenous, intramuscular, subcutaneous, intrathecal,intracavernosal or intraperitoneal injection or intravenous infusion),for topical administration, for sublingual administration, foradministration by inhalation, by a skin patch, by an implant, by asuppository, for transdermal, nasal, intravitreal, rectal or vaginaladministration, and the like. Such suitable administration forms —whichmay be solid, semi-solid or liquid, depending on the manner ofadministration —as well as methods and carriers for use in thepreparation thereof, will be clear to the skilled person.

Pharmaceutical preparations for parenteral administration, such asintravenous, intramuscular, subcutaneous injection or intravenousinfusion may for example be sterile solutions, suspensions, dispersions,emulsions, or powders which comprise the active ingredient and which aresuitable, optionally after a further dissolution or dilution step, forinfusion or injection. Suitable carriers or diluents for suchpreparations for example include, without limitation, sterile water andpharmaceutically acceptable aqueous buffers and solutions such asphysiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution; water oils; glycerol; ethanol; glycolssuch as propylene glycol, as well as mineral oils, animal oils andvegetable oils, for example peanut oil, soybean oil, as well as suitablemixtures thereof.

Solutions of the active compound or its salts may also contain apreservative to prevent the growth of microorganisms, such asantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal (thiomersal), and thelike. In many cases, it will be preferable to include isotonic agents,for example, sugars, buffers or sodium chloride. The proper fluidity canbe maintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. Other agents delaying absorption, forexample, aluminum monostearate and gelatin, may also be added.

In all cases, the ultimate dosage form must be sterile, fluid and stableunder the conditions of manufacture and storage. Sterile injectablesolutions are prepared by incorporating the active compound in therequired amount in the appropriate solvent with various of the otheringredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze drying techniques, which yield a powder ofthe active ingredient plus any additional desired ingredient present inthe previously sterile-filtered solutions.

Usually, aqueous solutions or suspensions will be preferred. Generally,suitable formulations for therapeutic proteins such as the polypeptidesof the invention are buffered protein solutions, such as solutionsincluding the protein in a suitable concentration (such as from 0.001 to400 mg/ml, preferably from 0.005 to 200 mg/ml, more preferably 0.01 to200 mg/ml, more preferably 1.0-100 mg/ml, such as 1.0 mg/ml (i.v.administration) or 100 mg/ml (s.c. administration) and an aqueous buffersuch as:

-   -   phosphate buffered saline, pH 7.4,    -   other phosphate buffers, pH 6.2 to 8.2,    -   histidine buffers, pH 5.5 to 7.0,    -   succinate buffers, pH 3.2 to 6.6, and    -   citrate buffers, pH 2.1 to 6.2,        and, optionally, salts (e.g. NaCl) and/or sugars or polyalcohols        (such as trehalose, mannitol, or glycerol) for providing        isotonicity of the solution.

Preferred buffered protein solutions are solutions including about 0.05mg/ml of the polypeptide of the invention dissolved in 25 mM phosphatebuffer, pH 6.5, adjusted to isotonicity by adding 220 mM trehalose. Inaddition, other agents such as a detergent, e.g. 0.02% Tween-20 orTween-80, may be included in such solutions. Formulations forsubcutaneous application may include significantly higher concentrationsof the polypeptide of the invention, such as up to 100 mg/ml or evenabove 100 mg/ml. However, it will be clear to the person skilled in theart that the ingredients and the amounts thereof as given above do onlyrepresent one, preferred option. Alternatives and variations thereofwill be immediately apparent to the skilled person, or can easily beconceived starting from the above disclosure.

The polypeptides of the invention may also be administered usingsuitable depot, slow-release or sustained-release formulations, e.g.suitable for injection, using controlled-release devices forimplantation under the skin, and/or using a dosing pump or other devicesknown per se for the administration of pharmaceutically activesubstances or principles. In addition, the polypeptides of the inventionmay be formulated in the form of a gel, cream, spray, drop, patch orfilm which, if placed on the skin, passes through the skin.

Also, compared to conventional antibodies or antibody fragments, onemajor advantage of the use of the polypeptides of the invention is thatthey can also be easily administered via routes other than parenteraladministration and can be easily formulated for such administration. Forexample, as described in the international application WO2004/041867,such polypeptides may be formulated for oral, intranasal, intrapulmonaryand transdermal administration.

According to another embodiment of the invention there is provided apharmaceutical combination comprising at least one polypeptide of theinvention as disclosed herein and at least one other therapeutic agentselected from the group consisting of statins, antiplatelets,anticoagulants, antidiabetics and anti hypertensives.

Such pharmaceutical combination may optionally additionally comprise adiluent, excipient, adjuvant and/or stabilizer.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition. Also, when two ormore active substances or principles are to be used as part of acombined treatment regimen, each of the substances or principles may beadministered in the same amount and according to the same regimen asused when the compound or principle is used on its own, and suchcombined use may or may not lead to a synergistic effect. However, whenthe combined use of the two or more active substances or principlesleads to a synergistic effect, it may also be possible to reduce theamount of one, more or all of the substances or principles to beadministered, while still achieving the desired therapeutic action. Thismay for example be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

Yet, a further embodiment of the invention is a method for treating thediseases and disorders as set out above, comprising administering to anindividual, simultaneously, separately or sequentially, an effectiveamount of at least one polypeptide of the invention and at least oneagent selected from the group consisting of a statin, an antiplatelet,an anticoagulant, an antidiabetic and an antihypertensive.

According to a further aspect of the invention, the polypeptide of theinvention is prepared to be administered in combination with other drugsused for the treatment of the diseases and disorders set out above, suchother drugs being selected from the group consisting of a statin, anantiplatelet, an anticoagulant, an antidiabetic and an antihypertensive.

According to still another aspect of the invention, drugs used for thetreatment of the diseases and disorders set out above, such drugs beingselected from the group consisting of a statin, an antiplatelet, ananticoagulant, an antidiabetic and an antihypertensive are prepared tobe administered in combination with the polypeptide of the invention.

According to a further aspect of the invention, the polypeptide of theinvention is used in combination with a device useful for theadministration of the polypeptide, such as a syringe, injector pen, orother device.

According to still another embodiment of the invention, there isprovided a method of diagnosing a disease, disorder or conditionmediated by CX3CR1 dysfunction comprising the steps of:

-   a) obtaining a sample from a subject, and-   b) contacting, in vitro, the sample with a polypeptide of the    invention as defined above, and-   c) detecting the binding of said polypeptide to said sample, and-   d) comparing the binding detected in step (c) with a standard,    wherein a difference in binding relative to said sample is    diagnostic of a disease, disorder or condition characterised by    CX3CR1 dysfunction.

According to another embodiment of the invention, there is provided amethod of diagnosing a disease, disorder or condition mediated by CX3CR1dysfunction comprising the steps of:

-   a) obtaining a sample from a subject, and-   b) contacting the sample with a polypeptide of the invention as    defined above;-   c) determining the amount of CX3CR1 in the sample; and-   d) comparing the amount determined in step (c) with a standard,    wherein a difference in amount relative to said sample is diagnostic    of a disease, disorder or condition characterised by CX3CR1    dysfunction.

The above diagnostic methods can also be used for monitoring theeffectiveness of a therapeutic treatment of a subject.

According to another embodiment of the invention, there is provided akit for diagnosing a disease, disorder or condition mediated by CX3CR1dysfunction, for use in a method as defined above, such kit comprisingat least one polypeptide of the invention and, optionally, one or moremedia, detection means and/or in vitro or in vivo imaging agents, and,further optionally, instructions of use. Suitable in vivo imaging agentsinclude 99mTc, 111Indium, 123Iodine, and, for magnetic resonanceimaging, paramagnetic compounds.

The invention further provides a kit comprising at least one polypeptideof the invention and, additionally, one or more other componentsselected from the group consisting of other drugs used for the treatmentof the diseases and disorders as described above, and devices asdescribed above.

The invention further provides methods of manufacturing a polypeptide ofthe invention, such methods generally comprising the steps of:

-   -   culturing host cells comprising a nucleic acid capable of        encoding a polypeptide of the invention (hereinafter: “nucleic        acid of the invention”) under conditions that allow expression        of the polypeptide of the invention; and,    -   recovering or isolating the polypeptide expressed by the host        cells from the culture; and    -   optionally further purifying and/or modifying and/or formulating        the polypeptide of the invention.

A nucleic acid of the invention can be genomic DNA, cDNA or syntheticDNA (such as DNA with a codon usage that has been specifically adaptedfor expression in the intended host cell or host organism). According toone embodiment of the invention, the nucleic acid of the invention is inessentially isolated form, as defined hereabove.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a vector, such as for example a plasmid, cosmid orYAC, which again may be in essentially isolated form. The vector mayespecially be an expression vector, i.e. a vector that can provide forexpression of the polypeptide in vitro and/or in vivo (e.g. in asuitable host cell, host organism and/or expression system). Suchexpression vector generally comprises at least one nucleic acid of theinvention that is operably linked to one or more suitable regulatoryelement(s), such as promoter(s), enhancer(s), terminator(s), and thelike. Specific examples of such regulatory elements and other elements,such as integration factor(s), selection marker(s), signal or leadersequence(s), reporter gene(s), and the like, useful or necessary forexpressing polypeptides of the invention, are disclosed e.g. on pp. 131to 133 of WO2006/040153.

The nucleic acids of the invention can be prepared or obtained in amanner known per se (e.g. by automated DNA synthesis and/or recombinantDNA technology), based on the information on the amino acid sequencesfor the polypeptides of the invention given herein, and/or can beisolated from a suitable natural source.

According to another embodiment, the invention relates to a host or hostcell that expresses or is capable of expressing a polypeptide of theinvention; and/or that contains a nucleic acid encoding a polypeptide ofthe invention. According to a particularly preferred embodiment, saidhost cells are bacterial cells, yeast cells, fungal cells or mammaliancells.

Suitable bacterial cells include cells from gram-negative bacterialstrains such as strains of Escherichia coli, Proteus, and Pseudomonas,and gram-positive bacterial strains such as strains of Bacillus,Streptomyces, Staphylococcus, and Lactococcus. Suitable fungal cellinclude cells from species of Trichoderma, Neurospora, and Aspergillus.Suitable yeast cells include cells from species of Saccharomyces (forexample Saccharomyces cerevisiae), Schizosaccharomyces (for exampleSchizosaccharomyces pombe), Pichia (for example Pichia pastoris andPichia methanolica), and Hansenula.

Suitable mammalian cells include for example CHO cells, BHK cells, HeLacells, COS cells, NS0 cells, HEK cells, and the like. However, amphibiancells, insect cells, plant cells, and any other cells used in the artfor the expression of heterologous proteins can be used as well.

For production on industrial scale, preferred heterologous hosts for the(industrial) production of immunoglobulin single variable domainpolypeptides and protein therapeutics containing them include strains ofE. coli, Pichia pastoris, and S. cerevisiae that are suitable for largescale expression, production and fermentation, and in particular forlarge scale (bio-)pharmaceutical expression, production andfermentation.

The choice of the specific expression system would depend in part on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of a polypeptide of theinvention for which glycosylation is desired or required wouldnecessitate the use of mammalian expression hosts that have the abilityto glycosylate the expressed protein. In this respect, it will be clearto the skilled person that the glycosylation pattern obtained (i.e. thekind, number and position of residues attached) will depend on the cellor cell line that is used for the expression.

Polypeptides of the invention produced in a cell as set out above can beproduced either intracellullarly (e.g. in the cytosol, in the periplasmaor in inclusion bodies) and then isolated from the host cells andoptionally further purified; or they can be produced extracellularly(secreted into the medium in which the host cells are cultured) and thenisolated from the culture medium and optionally further purified.

Further methods and reagents used for the recombinant production ofpolypeptides, such as suitable expression vectors, transformation ortransfection methods, selection markers, methods of induction of proteinexpression, culture conditions, and the like, are known in the art.Similarly, protein isolation and purification techniques useful in amethod of manufacture of a polypeptide of the invention are well knownto the skilled person.

Production of the polypeptides of the invention through fermentation inconvenient recombinant host organisms such as E. coli and yeast iscost-effective, as compared to conventional antibodies which alsorequire expensive mammalian cell culture facilities. Furthermore,achievable levels of expression are high and yields of the polypeptidesof the invention are in the range of 1 to 10 g/l (E. coli) and up to 10g/l (yeast) and more.

EXAMPLES

Generation CHO, Baf/3, Caki and HEK293 cell lines overexpressing humanCX3CR1 or cynomolgus CX3CR1

CHO and Baf/3 cells overexpressing human or cynomolgus CX3CR1 weregenerated using techniques known in the art. Cells expressing human CCR2or CCR5 were also generated using techniques known in the art.

The cDNA was cloned into pCDNA3.1(+)-neo for human CX3CR1 whereaspcDNA-DEST40-neo was used for mouse CX3CR1.

The amino acid sequences of humanCX3CR1 and cynomolgus CX3CR1 aredepicted in SEQ ID NO: 255 and 256, respectively.

To establish Camel Kidney (Caki) cells overexpressing human CX3CR1 ormouse CX3CR1, parental Caki cells were electroporated withpCDNA3.1(+)-neo-hCX3CR1 or pcDNA-DEST40-neo-mCX3CR1, respectively. Forall conditions, transfectants were selected by adding 1 mg/mL geneticin(Invitrogen, Carlsbad, Calif., USA).

Human Embyonic Kidney (HEK293) cells overexpressing human CX3CR1 orcynomolgus CX3CR1 were generated by lipid-mediated transfection withFugene (Roche) of pCDNA3.1(+)-neo-hCX3CR1 or cyCX3CR1 plasmids,respectively, in the HEK293 parental cell line. These cells were used astransient transfectants and as such not put under selection. In brief,21 0E6 cells were seeded per T75 and incubated overnight beforetransfection. After removal of the culture medium, cells weretransfected with the respective plasmids (9 μg) and Fugene (27 μl)according to manufacturer's instructions. 48 hours post transfection,cells were harvested and frozen for further usage.

Example 1 Immunization with CX3CR1 Induces a Humoral Immune Response inIlama

1.1. Immunizations

After approval of the Ethical Committee (University Antwerp, Belgium,UA2008A1, 2008/096, 2007/068), 9 Ilamas (designated No. 368, 369, 370,381, 382, 384, 312, 313 and 314) were immunized.

Six Ilamas (312, 313, 314, 381, 382 and 384) were immunized with 4intramuscular injections (2 mg/dose at weekly or biweekly intervals) ofpVAX1-huCX3CR1 plasmid vector (Invitrogen, Carlsbad, Calif., USA). ThreeIlamas (381, 382 and 384) subsequently received 4 subcutaneousinjections of human CX3CR1 overexpressing Caki cells which wereestablished as described above. Cells were re-suspended in D-PBS andkept on ice prior to injection.

Three additional Ilamas (designated No. 368, 369 and 370) were immunizedaccording to standard protocols with 4 subcutaneous injections of humanCX3CR1 overexpressing Caki cells which were established as describedabove. Cells were re-suspended in D-PBS and kept on ice prior toinjection. Subsequently, these Ilamas were administered two injectionswith recombinant CX3CR1 NT/EC3 fragment coupled to BSA (Table 13).Peptides were ordered at NeoMPS (Polypeptidegroup, Strasbourg, France)and coupled to BSA according to standard protocols.

TABLE 13 Sequence of peptide fragments used for immunization boostSEQ ID Fragment sequence NO: CX3CR1-NTAc-Met-Asp-Gln-Phe-Pro-Glu-Ser-Val-Thr-Glu-Asn- 228Phe-Glu-Tyr-Asp-Asp-Leu-Ala-Glu-Ala-Cys-NH2 CX3CR1-Ac-Lys-Leu-Tyr-Asp-Phe-Phe-Pro-Ser-Cys-Asp- 229 EC3Met-Arg-Lys-Asp-Leu-Arg-Leu-NH2

The first injection was formulated in Complete Freund's Adjuvant (Difco,Detroit, Mich., USA), while the subsequent injection was formulated inIncomplete Freund's Adjuvant (Difco, Detroit, Mich., USA).

1.2. Evaluation of Induced Immune Responses in Ilama

To evaluate the induction of immune responses in the animals againsthuman CX3CR1 by ELISA or FACS, sera were collected from Ilamas 312,313and 314 at day 0 (pre-immune), and different time points in theimmunization schedule (time of peripheral blood lymphocyte [PBL]collection).

In short, Neutravidin (2 μg/ml) was immobilized overnight at 4° C. in a96-well Maxisorb plate (Nunc, Wiesbaden, Germany). Wells were blockedwith a casein solution (1%) in PBS. Subsequently biotinylatedrecombinant NT fragment (Polypeptide, Strasbourg, France) orbiotinylated EC3 fragments of CX3CR1 (Polypeptide, Strasbourg, France)were captured at 2 μg/ml. After addition of serum dilutions,specifically bound immunoglobulins were detected using a horseradishperoxidase (HRP)-conjugated goat anti-Ilama immunoglobulin (BethylLaboratories Inc., Montgomery, Tex., USA) and a subsequent enzymaticreaction in the presence of the substrate TMB One(3,3′,5,5′-tetramentylbenzidine) (Promega, Mannheim, Germany), showingthat a significant antibody-dependent immune response against CX3CR1 wasinduced after the peptide immunizations.

Additionally, serum titers of cell immunized animals were confirmed byFACS analysis on actively growing human CX3CR1 overexpressing CHO cells.The CX3CR1 serum titer responses for Ilamas 368, 369 and 370 weredetermined with serum sampled after 4 cell immunizations (day 49), 4cell immunizations and 1 peptide boost (day 77) and 4 cell immunizationsand 2 peptide boosts (day 81). Cells were harvested and washed beforeincubation with the serum dilutions. Detection was performed with goatanti-Ilama IgG (Bethyl, Montgomery, Tex., USA) followed by donkeyanti-goat coupled with PE (Jackson Laboratories, Suffolk, UK) and readout by analysis on FACSArray (BD Biosciences). A summary of the obtainedserum responses as determined by either ELISA or FACS is shown in Table14 and Table 15.

TABLE 14 Serum titer analysis for the cell/peptide immunized animalsELISA FACS Recombi- Recom- After nant binant After cell peptide LlamaImmunogen NT EC3 immunization boosts 368 Caki- + +/− − − huCX3CR1 +NT/EC3 peptide 369 Caki- + +/− + + huCX3CR1 + NT/EC3 peptide 370 Caki-++ ++ − + huCX3CR1 + NT/EC3 peptide

TABLE 15 Serum titer analysis for the DNA/cell immunized animals ELISAFACS Recombi- Recom- After nant binant After DNA Cell Llama Immunogen NTEC3 immunization boosts 381 DNA + Caki- ++ + ++ ++ huCX3CR1 382 DNA +Caki- + − − − huCX3CR1 384 DNA + Caki- ++ − − − huCX3CR1

For the DNA only immunized Ilamas (312, 313 and 314) no serum titer wasdetermined.

Example 2 Cloning of the Heavy-chain Only Antibody Fragment Repertoiresand Preparation of Phage

Following the final immunogen injection of each subset, immune tissuesas the source of B-cells that produce the heavy-chain antibodies werecollected from the immunized Ilamas. For Ilama 312,313 and 314, two150-ml blood samples, collected 4 and 8 days after the last antigeninjection were collected per animal. For Ilamas 368, 369 and 370 four150 ml blood samples were collected, 5 and 7 days after the last cellimmunization and additionally 4 and 8 days after the last peptideimmunization. Next to those, two lymph node biopsies were taken, 12 daysafter the last cell immunization and 12 days after the last peptideimmunization. For Ilamas 381, 382 and 384 five 150 ml blood samples werecollected, 8 days after the last DNA immunization and additionally 4days after the first cell boost, 8 and 11 days after the second cellboost and 8 days after the last cell immunization. Next to those, onelymph node biopsy was taken, 8 days after the second cell immunization.

From the blood samples, peripheral blood lymphocytes (PBLs) wereprepared using Ficoll-Hypaque according to the manufacturer'sinstructions (Amersham Biosciences, Piscataway, N.J., USA). From thePBLs and the lymph node biopsy (LN), total RNA was extracted, which wasused as starting material for RT-PCR to amplify the VHH encoding DNAsegments.

For each immunized Ilama, libraries were constructed by pooling thetotal RNA isolated from samples originating from a certain subset of theimmunization schedule i.e. after one type of immunization antigen, andfor some Ilamas samples from the different animals were pooled into onelibrary (Table 16).

TABLE 16 Pooling of the different sample for library constructionLibrary Name Llama Sample 368-PBL1 + 2 + LN-V-100209 368 PBL 1 and 2, LN369 + 370-PBL1 + 2 + LN-V-100209 369, 370 PBL 1 and 2, LN 368-PBL3 +4-V-280909 368 PBL 3 and 4 369-PBL3 + 4-V-070409 369 PBL 3 and 4370-PBL3 + 4-V-070409 370 PBL 3 and 4 381-PBL1-V-180310 381 PBL 1382-PBL1-V-180310 382 PBL1 384-PBL1-V-180310 384 PBL1 381-PBL1 + 2 + 3 +4 + 5 + LN-V-280909 381 PBL 1, 2, 3, 4, 5 and LN 382-PBL1 + 2 + 3 + 4 +5 + LN-V-280909 382 PBL 1, 2, 3, 4, 5 and LN 384-PBL1 + 2 + 3 + 4 +5 +Ln-V-280909 384 PBL 1, 2, 3, 4, 5 and LN 312 + 313 + 314-PBL1 +2-V-220210 312, 313 and 314 PBL 1 and 2 312-PBL1 + 2-V-180310 312 PBL 1and 2 313-PBL1 + 2-V-180310 313 PBL 1 and 2 314-PBL1 + 2-V-180310 314PBL 1 and 2

In short, the PCR-amplified VHH repertoire was cloned via specificrestriction sites into a vector designed to facilitate phage display ofthe VHH library. The vector was derived from pUC119 and contains theLacZ promoter, a M13 phage gill protein coding sequence, a resistancegene for ampicillin or carbenicillin, a multiple cloning site and ahybrid gIII-pelB leader sequence (pAX050). In frame with the VHH codingsequence, the vector encodes a C-terminal c-myc tag and a His6 tag.Phage were prepared according to standard protocols and stored afterfilter sterilization at 4° C. or at −80° C. in 20% glycerol for furtheruse.

Example 3 Selection of CX3CR1 specific VHHs via Phage Display

VHH repertoires obtained from all Ilamas and cloned as phage librarywere used in different selection strategies, applying a multiplicity ofselection conditions. Variables include i) the presentation form of theCX3CR1 protein (on different cell backgrounds or on liposomes/VLPs), ii)the antigen presentation method (In solution when using cells or coatedonto plates when using VLPs), iii) the antigen concentration iv) theorthologue used (human or cynomolgus) v) the number of selection roundsand vi) different elution methods (non-specific via trypsin or specificvia the ligand Fractalkine). All solid coated phase selections were donein Maxisorp 96-well plates (Nunc, Wiesbaden, Germany).

Selections were performed as follows: CX3CR1 antigen preparations forsolid and solution phase selection formats were presented as describedabove at multiple concentrations. After 2h incubation with the phagelibraries followed by extensive washing, bound phages were eluted withtrypsin (1 mg/mL) for 15 minutes. When trypsin was used for phageelution, the protease activity was immediately neutralized by applying0.8 mM protease inhibitor ABSF. As control, selections without antigenwere performed in parallel.

Phage outputs were used to infect E. coli which were then in turn usedto prepare phage for the next selection round (phage rescue) After thesecond round selection the phage outputs were used to infect E. coliwhich were then plated on agar plates (LB+carb+glucose^(2%)) foranalysis of individual VHH clones. In order to screen a selection outputfor specific binders, single colonies were picked from the agar platesand grown in 1 mL 96-deep-well plates. LacZ-controlled VHH expressionwas induced by adding IPTG (1mM final) in the absence of glucose.Periplasmic extracts (in a volume of ˜80 uL) were prepared according tostandard protocols.

Example 4 Screening of Periplasmic Extracts in CX3CR1-FraktalkineCompetition FACS Assay

Periplasmic extracts were screened in a human CX3CR1/human FractalkineFACS competition assay to assess the blocking capacity of the expressedVHHs. Human CX3CR1 was presented on CHO cells overexpressing CX3CR1.Both a setup using cells harvested from an actively growing culture anda setup using frozen cells was used. As a detection reagent labeledfractalkine was used (R&D Systems, Minneapolis, Minn., USA) labeled withalexa647 (A647-Fractalkine) at a degree of labeling of 1. To setup theassay, first a titration series of the labeled fractalkine was performedon the CHO-huCX3CR1 cells in order to determine the EC50 value forbinding. Initially screening was performed at a higher concentration offractalkine (3 nM) to increase the assay robustness. To increase thesensitivity of the screening to a maximum, the EC30 concentration (1 nM)was chosen for subsequent screening. In brief 50 μl of periplasmicextract was added to 6 nM labeled fractalkine (50 μ;) and 200 000CHO-huCX3CR1 cells. After one hour incubation at 4 C, cells were washedthree times before read out was performed on a FACS Array (BectonDickinson). First a gate was set on the intact cells as determined fromthe scatter profile. Next, dead cells were gated out by theirfluorescence profile from the PI stain (Sigma, St Louis, US). Thefluorescence profile from the alexa647 label was determined for eachsample and used for calculation of blocking capacity. As controls,conditions were taken along where there was no VHH present in the periextract or a known irrelevant VHH and samples were included where excesscold fractalkine was included. For each sample the percentage block wasdetermined using the control samples to determine the assay window.

From this screening, VHHs were selected and sequence analysis revealed120 unique VHHs belonging to 3 different B-cell lineages. The totalnumber of variants found for each B-cell lineage is depicted in Table17.

TABLE 17 Selection parameters used for the identification of thehumanCX3CR1 specific VHH B-cell lineages B-cell Representative # lineageVHH ID variants libraries 9 CX3CR1BII11H11 4 368-PBL1 + 2 + LN-V-10020913 CX3CR1BII18E06 68 368-PBL1 + 2 + LN-V-100209 368-PBL3 + 4-V-280909101 CX3CR1BII66B02 48 312 + 313 + 314-PBL1 + 2-V-220210 314-PBL1 +2-V-180310

An overview of the selection procedure and performance during initialscreening is given for all VHHs in Table 18.

TABLE 18 Selection conditions and primary screening result for thehuCX3CR1 specific VHH Selections VHH ID Family Library first roundsecond round % block CX3CR1BII 9 368- BA/F3_hCX3CR1 total CHO- total99.0 PMP11H11 PBL1+2+LN- (trypsin) K1_hCX3CR1 (trypsin) V-100209CX3CR1BII 13 368- BA/F3_hCX3CR1 total CHO- total 53.1 PMP18E6 PBL3+4-V-(trypsin) K1_hCX3CR1 (trypsin) 280909 CX3CR1BII 101 312+313+314- VLPs-hFrac (2 VLPs- hFrac (2 93.8 PMP54A12 PBL1+2- hCX3CR1 μM) hCX3CR1 μM)V-220210 (10 U) (10 U) CX3CR1BII 101 312+313+314- VLPs- hFrac (2 VLPs-hFrac (2 90.8 PMP54A3 PBL1+2- hCX3CR1 μM) hCX3CR1 μM) V-220210 (10 U)(10 U) CX3CR1BII 101 312+313+314- VLPs- hFrac (2 VLPs- hFrac (2 86.6PMP54A4 PBL1+2- hCX3CR1 μM) hCX3CR1 μM) V-220210 (10 U) (10 U) CX3CR1BII101 312+313+314- VLPs- hFrac (2 VLPs- hFrac (2 92.5 PMP54A5 PBL1+2-hCX3CR1 μM) hCX3CR1 μM) V-220210 (10 U) (10 U) CX3CR1BII 101312+313+314- VLPs- total VLPs- total 68.9 PMP54A7 PBL1+2- hCX3CR1(trypsin) hCX3CR1 (trypsin) V-220210 (10 U) (10 U) CX3CR1BII 101312+313+314- VLPs- hFrac (2 VLPs- hFrac (2 92.1 PMP54B1 PBL1+2- hCX3CR1μM) hCX3CR1 μM) V-220210 (10 U) (10 U) CX3CR1BII 101 312+313+314- VLPs-hFrac (2 VLPs- hFrac (2 65.3 PMP54B2 PBL1+2- hCX3CR1 μM) hCX3CR1 μM)V-220210 (10 U) (10 U) CX3CR1BII 101 312+313+314- VLPs- hFrac (2 VLPs-hFrac (2 90.1 PMP54B3 PBL1+2- hCX3CR1 μM) hCX3CR1 μM) V-220210 (10 U)(10 U) CX3CR1BII 101 312+313+314- VLPs- hFrac (2 VLPs- hFrac (2 92.6PMP54B5 PBL1+2- hCX3CR1 μM) hCX3CR1 μM) V-220210 (10 U) (10 U) CX3CR1BII101 312+313+314- VLPs- hFrac (2 VLPs- hFrac (2 87.8 PMP54D5 PBL1+2-hCX3CR1 μM) hCX3CR1 μM) V-220210 (10 U) (10 U) CX3CR1BII 101312+313+314- VLPs- total VLPs- total 64.1 PMP54D8 PBL1+2- hCX3CR1(trypsin) hCX3CR1 (trypsin) V-220210 (10 U) (10 U) CX3CR1BII 101312+313+314- VLPs- hFrac (2 VLPs- hFrac (2 96.6 PMP54F6 PBL1+2- hCX3CR1μM) hCX3CR1 μM) V-220210 (10 U) (10 U) CX3CR1BII 101 312+313+314- VLPs-hFrac (2 VLPs- hFrac (2 74.7 PMP54G3 PBL1+2- hCX3CR1 μM) hCX3CR1 μM)V-220210 (10 U) (10 U) CX3CR1BII 101 312+313+314- VLPs- hFrac (2 VLPs-hFrac (2 74.6 PMP54H1 PBL1+2- hCX3CR1 μM) hCX3CR1 μM) V-220210 (10 U)(10 U) CX3CR1BII 101 312+313+314- VLPs- hFrac (2 VLPs- hFrac (2 96.0PMP54H4 PBL1+2- hCX3CR1 μM) hCX3CR1 μM) V-220210 (10 U) (10 U) CX3CR1BII101 314- VLPs- hFrac (2 VLPs- hFrac (2 73.5 PMP61F10 PBL1+2-V- hCX3CR1μM) hCX3CR1 μM) 180310 (10 U) (10 U) CX3CR1BII 101 314- VLPs- hFrac (2VLPs- hFrac (2 68.4 PMP61D1 PBL1+2-V- hCX3CR1 μM) hCX3CR1 μM) 180310 (10U) (10 U) CX3CR1BII 101 314- VLPs- hFrac (2 VLPs- hFrac (2 94.9 PMP61D5PBL1+2-V- hCX3CR1 μM) hCX3CR1 μM) 180310 (10 U) (10 U) CX3CR1BII 101314- VLPs- hFrac (2 VLPs- hFrac (2 70.3 PMP61E2 PBL1+2-V- hCX3CR1 μM)hCX3CR1 μM) 180310 (10 U) (10 U) CX3CR1BII 101 314- VLPs- hFrac (2 VLPs-hFrac (2 96.5 PMP61F11 PBL1+2-V- hCX3CR1 μM) hCX3CR1 μM) 180310 (10 U)(10 U) CX3CR1BII 101 314- VLPs- hFrac (2 VLPs- hFrac (2 82.0 PMP61G2PBL1+2-V- hCX3CR1 μM) hCX3CR1 μM) 180310 (10 U) (10 U) CX3CR1BII 101314- VLPs- hFrac (2 VLPs- hFrac (2 92.1 PMP61G3 PBL1+2-V- hCX3CR1 μM)hCX3CR1 μM) 180310 (10 U) (10 U) CX3CR1BII 101 314- VLPs- hFrac (2 VLPs-hFrac (2 94.5 PMP61G4 PBL1+2-V- hCX3CR1 μM) hCX3CR1 μM) 180310 (10 U)(10 U) CX3CR1BII 101 314- VLPs- hFrac (2 VLPs- hFrac (2 94.4 PMP61F4PBL1+2-V- hCX3CR1 μM) hCX3CR1 μM) 180310 (10 U) (10 U) CX3CR1BII 101314- VLPs- hFrac (2 VLPs- hFrac (2 78.0 PMP61A11 PBL1+2-V- hCX3CR1 μM)hCX3CR1 μM) 180310 (10 U) (10 U) CX3CR1BII 101 314- VLPs- hFrac (2 VLPs-hFrac (2 94.5 PMP61B2 PBL1+2-V- hCX3CR1 μM) hCX3CR1 μM) 180310 (10 U)(10 U) CX3CR1BII 101 314- VLPs- total VLPs- total 69.4 PMP61C9 PBL1+2-V-hCX3CR1 (trypsin) hCX3CR1 (trypsin) 180310 (10 U) (10 U) CX3CR1BII 101312+313+314- VLPs- hFrac (2 VLPs- hFrac (2 #N/A PMP65H02 PBL1+2- hCX3CR1μM) hCX3CR1 μM) V-220210 (10 U) (10 U) CX3CR1BII 101 312+313+314- VLPs-hFrac (2 VLPs- hFrac (2 #N/A PMP65E11 PBL1+2- hCX3CR1 μM) hCX3CR1 μM)V-220210 (10 U) (10 U) CX3CR1BII 101 312+313+314- VLPs- hFrac (2 VLPs-hFrac (2 #N/A PMP65E10 PBL1+2- hCX3CR1 μM) hCX3CR1 μM) V-220210 (10 U)(10 U) CX3CR1BII 101 312+313+314- VLPs- hFrac (2 VLPs- hFrac (2 #N/APMP65E05 PBL1+2- hCX3CR1 μM) hCX3CR1 μM) V-220210 (10 U) (10 U)CX3CR1BII 101 312+313+314- VLPs- hFrac (2 VLPs- hFrac (2 #N/A PMP65B11PBL1+2- hCX3CR1 μM) hCX3CR1 μM) V-220210 (10 U) (10 U) CX3CR1BII 101312+313+314- VLPs- hFrac (2 VLPs- hFrac (2 #N/A PMP65B07 PBL1+2- hCX3CR1μM) hCX3CR1 μM) V-220210 (10 U) (10 U) CX3CR1BII 101 312+313+314- VLPs-hFrac (2 VLPs- hFrac (2 #N/A PMP65B09 PBL1+2- hCX3CR1 μM) hCX3CR1 μM)V-220210 (10 U) (10 U) CX3CR1BII 101 312+313+314- VLPs- hFrac (2 VLPs-hFrac (2 #N/A PMP65H01 PBL1+2- hCX3CR1 μM) hCX3CR1 μM) V-220210 (10 U)(10 U) CX3CR1BII 101 312+313+314- VLPs- hFrac (2 VLPs- hFrac (2 #N/APMP65G07 PBL1+2- hCX3CR1 μM) hCX3CR1 μM) V-220210 (10 U) (10 U)CX3CR1BII 101 314- VLPs- hFrac (2 VLPs- hFrac (2 #N/A PMP66H08 PBL1+2-V-hCX3CR1 μM) hCX3CR1 μM) 180310 (10 U) (10 U) CX3CR1BII 101 314- VLPs-hFrac (2 VLPs- hFrac (2 #N/A PMP66H04 PBL1+2-V- hCX3CR1 μM) hCX3CR1 μM)180310 (10 U) (10 U) CX3CR1BII 101 314- VLPs- hFrac (2 VLPs- hFrac (2#N/A PMP66F02 PBL1+2-V- hCX3CR1 μM) hCX3CR1 μM) 180310 (10 U) (10 U)CX3CR1BII 101 314- VLPs- hFrac (2 VLPs- hFrac (2 #N/A PMP66E11 PBL1+2-V-hCX3CR1 μM) hCX3CR1 μM) 180310 (10 U) (10 U) CX3CR1BII 101 314- VLPs-hFrac (2 VLPs- hFrac (2 #N/A PMP66D10 PBL1+2-V- hCX3CR1 μM) hCX3CR1 μM)180310 (10 U) (10 U) CX3CR1BII 101 314- VLPs- hFrac (2 VLPs- hFrac (2#N/A PMP66D08 PBL1+2-V- hCX3CR1 μM) hCX3CR1 μM) 180310 (10 U) (10 U)CX3CR1BII 101 314- VLPs- hFrac (2 VLPs- hFrac (2 #N/A PMP66B02 PBL1+2-V-hCX3CR1 μM) hCX3CR1 μM) 180310 (10 U) (10 U) CX3CR1BII 101 314- VLPs-hFrac (2 VLPs- hFrac (2 #N/A PMP66A04 PBL1+2-V- hCX3CR1 μM) hCX3CR1 μM)180310 (10 U) (10 U) CX3CR1BII 101 314- VLPs- hFrac (2 VLPs- hFrac (2#N/A PMP66D04 PBL1+2-V- hCX3CR1 μM) hCX3CR1 μM) 180310 (10 U) (10 U)CX3CR1BII 101 314- VLPs- hFrac (2 VLPs- hFrac (2 #N/A PMP66D02 PBL1+2-V-hCX3CR1 μM) hCX3CR1 μM) 180310 (10 U) (10 U) CX3CR1BII 101 314- VLPs-hFrac (2 VLPs- hFrac (2 #N/A PMP66D06 PBL1+2-V- hCX3CR1 μM) hCX3CR1 μM)180310 (10 U) (10 U) CX3CR1BII 101 314- VLPs- hFrac (2 VLPs- hFrac (2#N/A PMP66G01 PBL1+2-V- hCX3CR1 μM) hCX3CR1 μM) 180310 (10 U) (10 U)

The amino acid sequences of all obtained unique VHHs are shown in theSequence Listing and above (CDRs and framework regions were indicated).

Example 5 Characterization of Purified VHHs

Inhibitory anti-CX3CR1 VHHs selected from the screening described inExample 4 were further purified and characterized. Selected VHHs wereexpressed in E. coli TG1 as c-myc, His6-tagged proteins. Expression wasinduced by addition of 1 mM IPTG and allowed to continue for 4 hours at3TC. After spinning the cell cultures, periplasmic extracts wereprepared by freeze-thawing the pellets. These extracts were used asstarting material and VHHs were purified via IMAC and size exclusionchromatography (SEC) resulting in 95% purity as assessed via SDS-PAGE.

Inhibition by Anti-CX3CR1 VHHs of Human Fractalkine Binding to HumanCX3CR 1 Expressed on the BA/F3 Cells

The blocking capacity towards the ligand fractalkine of the VHHs wasevaluated in a human CX3CR1 competition FACS as outlined in Example 4.Either CHO-huCX3CR1 cells, BA/F3-huCX3CR1 cells or transientlytransfected HEK293T cells were used. The amount of labeled ligand usedin the different competition setups was also varied. The IC₅₀ values forVHHs blocking the interaction of human fractalkine to human CX3CR1 aredepicted in Table 19.

TABLE 19 Potency and efficacy of the VHH in a ligand competition FACSVHH ID Family Cell line IC50 % block Repeats 11H11 9 CHO-huCX3CR1 1.7E−8100 4 18E06 13 CHO-huCX3CR1 1.8E−9 33 4 54A12 101 CHO-huCX3CR1 2.1E−9104 2 54D08 101 CHO-huCX3CR1 1.5E−8 101 2 54A07 101 CHO-huCX3CR1 1.1E−878 2 54D05 101 CHO-huCX3CR1 2.7E−9 102 2 54B03 101 CHO-huCX3CR1 2.5E−8108 1 54G03 101 CHO-huCX3CR1 5.6E−8 107 1 11H11 9 BA/F3-huCX3CR1 8.1E−9100 3 18E06 13 BA/F3-huCX3CR1 2.8E−9 71 3 54A12 101 BA/F3-huCX3CR14.0E−9 100 4 54D08 101 BA/F3-huCX3CR1 3.8E−8 99 1 54A07 101BA/F3-huCX3CR1 1.5E−8 81 4 54D05 101 BA/F3-huCX3CR1 5.5E−9 99 1 54B03101 BA/F3-huCX3CR1 3.3E−8 99 1 54G03 101 BA/F3-huCX3CR1 9.8E−8 98 154A12 101 HEK293-huCX3CR1 6.8E−9 96 5 54D08 101 HEK293-huCX3CR1 8.4E−895 2 54A07 101 HEK293-huCX3CR1 2.3E−8 52 2 54D05 101 HEK293-huCX3CR15.3E−9 94 5 54B03 101 HEK293-huCX3CR1 6.7E−8 92 2 54G03 101HEK293-huCX3CR1 2.7E−7 89 2 61E02 101 HEK293-huCX3CR1 8.2E−8 98 2 61B04101 HEK293-huCX3CR1 5.7E−8 97 2 61B02 101 HEK293-huCX3CR1 1.0E−8 94 254H01 101 HEK293-huCX3CR1 1.0E−8 64 2 54A04 101 HEK293-huCX3CR1 4.9E−8100 2 61F11 101 HEK293-huCX3CR1 4.6E−8 96 2 61G03 101 HEK293-huCX3CR16.0E−8 96 2 61G04 101 HEK293-huCX3CR1 4.2E−8 96 2 66B02 101HEK293-huCX3CR1 2.5E−9 102 2 66G01 101 HEK293-huCX3CR1 1.4E−8 99 2

Inhibition by Anti-CX3CR1 VHHs of Human Fractalkine Induced C hemotaxisof BA/F3 Cells Overexpressing Human CX3CR1

To evaluate inhibition of Fractalkine induced chemotaxis, a chemotaxisassay was setup using the ChemoTx disposable chamber with 5 μm poresize(Neuroprobe, Gaithersburg, US). Cells were harvested from an activelygrowing culture and washed before use in assay medium, RPMI (Gibco,Carlsbad, US) supplemented with 0.1% BSA. The bottom chamber was filledwith 320 pM human Fractalkine in a total volume of 300 μl. Uponapplication of the membrane, 0.13E6 cells were deposited on top of themembrane in a total volume of 70 μl. Chemotaxis was allowed for 3 hoursat 37° C. in a humidified chamber with CO2. After this incubationperiod, the membrane was removed and cells in the bottom chamber wereresuspended. The amount of ATP present in the wells was determined usingthe CellTiter-Glo kit (Promega, Madison Wis., US). Read out wasperformed on an Envision (Perkin Elmer, Mass., US) with the standardsettings for luminescence read out. Titration series were performed intriplicate and each plate contained control samples in triplicate aswell. As control, a sample without VHH was included as well as a samplewhere no human Fractalkine was added to the bottom chamber. A summary ofthe results is shown in Table 20.

TABLE 20 Potency and efficacy of the VHH in blocking the fractalkineinduced chemotaxis VHH ID Fam IC50 % block Repeats 11H11 9 2E−7 89 518E06 13 NA 17 4 54A12 101 8E−8 84 6 54D08 101 NA 33 2 54A07 101 5E−8 454 54D05 101 7E−8 81 4 54B03 101 1E−7 73 1 54G03 101 NA 40 1 66B02 1012E−8 87 2 66G01 101 4E−7 54 2

Evaluation of the Cross Reactivity of the Anti-CX3CR1 VHHs AgainstCynomolgus CX3CR1

Initially, a FACS based binding setup was used to evaluate thecynomolgus cross reactivity. For this, the VHHs were incubated with therespective cells for 30 minutes at 4° C. followed by three wash stepsand subsequently incubated with the detection reagents. As detection, amouse anti-cmyc antibody (Serotec, MCA2200) followed by a goatanti-mouse antibody coupled to PE (Jackson 115-116-071) was used, eachincubation for 30 minutes at 4° C., followed by three wash steps.Results of the assay are shown in Table 21.

TABLE 21 EC50 value for binding of the respective VHH on human CX3CR1 oron cynomolgus CX3CR1 EC50 (M) EC50 (M) VHH ID Family Human Cynomolgusratio Repeats 11H11 9 8.0E−10 NA NA 2 18E06 13 1.5E−9 1.4E−8 9 2 54A12101 3.5E−8 1.1E−7 3.3 1 54A07 101 5.4E−7 3.0E−8 0.1 1 54D05 101 8.4E−105.0E−8 59.6 1

For later identified VHHs, a human Fractalkine competition FACS was setup using human or cynomolgus CX3CR1 expressed in HEK293T cells. Both thehuman and the cynomolgus receptor was transiently transfected in HEK293Tcells and transfections were matched by the binding of the labeledligand, human fractalkine. The competition was evaluated using the EC30concentration of fractalkine and as such obtained IC50 values are a goodestimate of the Ki value, a measure for affinity (Table 22). Theexperiment was performed as described in Example 4. The ratio of theIC50 values on cynomolgus monkey and human CX3CR1 was used to evaluatepotential differences in affinity for CX3CR1 in both species.

TABLE 22 Efficacy and potency of VHHs in ligand competition FACS towardshuman and cynomolgus CX3CR1 Human Cynomolgus IC50 % IC50 % VHH ID Family(M) block (M) block ratio Repeats 54A12 101 6.8E−9 96 6.7E−8 94 9.85 554D08 101 8.4E−8 95 7.2E−8 91 0.85 2 54A07 101 2.3E−8 52 2.8E−7 69 12.32 54D05 101 5.3E−9 94 6.4E−8 91 12.25 5 54B03 101 6.7E−8 92 4.7E−7 957.05 2 54G03 101 2.7E−7 89 4.7E−7 89 1.74 2 61E02 101 8.2E−8 98 4.6E−896 0.55 2 61B04 101 5.7E−8 97 8.7E−8 93 1.53 2 61B02 101 1.0E−8 944.5E−8 92 4.37 2 54H01 101 1.0E−8 64 6.8E−8 92 6.48 2 54A04 101 4.9E−8100 3.1E−8 91 0.64 2 61F11 101 4.6E−8 96 1.6E−7 95 3.58 2 61G03 1016.0E−8 96 3.0E−7 89 5.02 2 61G04 101 4.2E−8 96 2.0E−7 100 4.8 2 66B02101 2.5E−9 102 1.9E−8 97 7.5 2 66G01 101 1.4E−8 99 1.2E−7 100 8.29 2

Binding of the Anti-Human CX3CR 1 VHHs to Human CCR2, Human CCR5 orMouse CX3CR1

Specificity for the huCX3CR1 receptor was evaluated by performing a FACSbinding experiment on CHO-K1 parental cells or CHO cells expressinghuCCR2, huCCR5 or msCX3CR1. The VHHs were incubated with the respectivecell lines for 30 minutes at 4° C. followed by three wash steps andsubsequently incubated with the detection reagents. As detection, amouse anti-cmyc antibody (Serotec, MCA2200) followed by a goatanti-mouse antibody coupled to PE (Jackson 115-116-071) was used, eachincubation for 30 minutes at 4° C., followed by three wash steps. Foreach cell line a quality control with receptor-specific antibody wasincluded. In addition, the highest concentration of each VHH was alsoincubated with CHO cells expressing huCX3CR1 as a positive control. Nobinding to msCX3CR1, huCCR2 or huCCR5 could be observed.

Determination of the Epitope Bin

A competitive binding experiment was setup in order to determine whetherthe VHHs bind overlapping epitopes on CX3CR1. For this, the VHH 66B02labeled with alexa647 was used in a competition FACS on the BA/F3 cellsexpressing huCX3CR1. Representative VHHs from the three functionalfamilies were used as competitors for the binding of the labeled 66B02.The obtained IC50 values are shown in Table 23.

TABLE 23 Competition FACS based epitope binning VHH ID family IC50 (M) %block 11H11 9 4.9E−09 100 18E06 13 2.3E−09 100 66B02 101 1.5E−09 100

As a complete inhibition of 66B02 binding could be obtained by allrepresentative VHHs from the different ligand blocking families, it canbe concluded that all functional families bind in close enough proximityof each other such that they compete with binding of 66B02.

Example 6 Formatting of VHHs to Bivalency

Construction of Bivalents

In order to increase potency and/or efficacy from a selection of theobtained VHHs, bivalent molecules were constructed by geneticengineering. Two VHHs were genetically linked together with a 35GSlinker in between the two building blocks and subsequently expressed inE. coli as described above for the monovalent VHHs. Different bivalentconstructs were made as listed in Table 24.

TABLE 24 Representative bivalent formats Construct ID VHH identityFamily Linker VHH identity Family CX3CR1BII007 CX3CR1BII11H11 9 35GSCX3CR1BII18E6 13 CX3CR1BII009 CX3CR1BII18E6 13 35GS CX3CR1BII11H11 9CX3CR1BII012 CX3CR1BII54D08 101 35GS CX3CR1BII18E06 101 CX3CR1BII016CX3CR1BII54A12 101 35GS CX3CR1BII54A12 101 CX3CR1BII017 CX3CR1BII54D5101 35GS CX3CR1BII54D5 101 CX3CR1BII018 CX3CR1BII66B02 101 35GSCX3CR1BII66B02 101 CX3CR1BII019 CX3CR1BII66G01 101 35GS CX3CR1BII66G01101 CX3CR1BII020 CX3CR1BII54B5 101 35GS CX3CR1BII54B5 101 CX3CR1BII026CX3CR1BII11H11 9 35GS CX3CR1BII66B02 101 CX3CR1BII027 CX3CR1BII11H11 935GS CX3CR1BII54B5 101

Inhibition by Anti-CX3CR1 VHHs of Human Fractalkine Binding to HumanCX3CR 1 Expressed on the BA/F3 Cells

The inhibition of ligand binding to human CX3CR1 was investigated forthe different formats as described in Example 4. For thischaracterization the BA/F3-huCX3CR1 cell line was used showing stableexpression of the human CX3CR1 receptor. The alexa647 labeled ligandfractalkine was used at its EC30 concentration and thereby obtained IC50values are reflective of the Ki values. An overview of the obtained datais shown in Table 25.

TABLE 25 Potency of bivalent formats in ligand competition Construct IDCell line IC50 (M) % block Repeats CX3CR1BII007 CHO-huCX3CR1 3.8E−10 1002 CX3CR1BII009 CHO-huCX3CR1 7.0E−10 91 2 CX3CR1BII012 CHO-huCX3CR18.0E−10 93 1 CX3CR1BII016 HEK293-huCX3CR1 1.9E−10 102 2 CX3CR1BII017HEK293-huCX3CR1 3.1E−10 99 2 CX3CR1BII018 HEK293-huCX3CR1 3.0E−10 102 2CX3CR1BII019 HEK293-huCX3CR1 2.9E−10 100 2 CX3CR1BII020 HEK293-huCX3CR12.2E−10 102 2 CX3CR1BII026 BA/F3-huCX3CR1 7.0E−10 100 3 CX3CR1BII027BA/F3-huCX3CR1 6.7E−10 100 3

Inhibition by Anti-CX3CR1 VHHs of Human Fractalkine Induced Chemotaxisof BA/F3 Cells Overexpressing Human CX3CR1

Similar to what was described for the monovalent anti-CX3CR1 VHHs, theinhibition of fractalkine induced chemotaxis on the BA/F3-huCX3CR1 cellswas evaluated for the bivalent constructs. An identical assay setup wasused as described above and the obtained results are summarized in Table26.

TABLE 26 Inhibition of fractalkine induced chemotaxis by bivalent VHHconstructs Construct ID Cell line IC50 (M) % block Repeats CX3CR1BII007BA/F3-huCX3CR1 4E−9 101 5 CX3CR1BII009 BA/F3-huCX3CR1 2E−8 79 5CX3CR1BII012 BA/F3-huCX3CR1 4E−9 78 1 CX3CR1BII016 BA/F3-huCX3CR1 2E−988 3 CX3CR1BII017 BA/F3-huCX3CR1 3E−9 89 3 CX3CR1BII018 BA/F3-huCX3CR16E−10 98 6 CX3CR1BII019 BA/F3-huCX3CR1 2E−9 85 3 CX3CR1BII020BA/F3-huCX3CR1 2E−9 85 3 CX3CR1BII026 BA/F3-huCX3CR1 3E−10 98 1CX3CR1BII027 BA/F3-huCX3CR1 9E−10 98 1

Evaluation of the Cross Reactivity of the Anti-CX3CR1 VHHs AgainstCynomolgus CX3CR1

Also for the bivalent constructs the cross reactivity towards cynomolgusCX3CR1 was evaluated and compared with the human reactivity. Asdescribed earlier, either a binding setup (Table 27) or a ligandcompetition setup (Table 28) were applied using transient transfectedHEK293T cells. Batches of transient transfected cells were matched bytheir receptor expression level.

TABLE 27 Binding of bivalent constructs to human or cynomolgus CX3CR1EC50 (M) EC50 (M) Construct ID Cell line Human Cynomolgus ratio RepeatsCX3CR1BII007 HEK293T 3.1E−10 4.8E−8 154 2 CX3CR1BII009 HEK293T 2.0E−96.8E−9 3.3 2 CX3CR1BII012 HEK293T 5.6E−11 6.3E−11 1.1 1

TABLE 28 Ligand competition of bivalent constructs on human orcynomolgus CX3CR1 Human Cynomolgus Construct ID Cell line IC50 (M) %block IC50 (M) % block ratio Repeats CX3CR1BII016 HEK293T 1.9E−10 1026.9E−10 98 3.67 2 CX3CR1BII017 HEK293T 3.1E−10 99 1.6E−9  95 5.34 2CX3CR1BII018 HEK293T 3.0E−11 102 1.0E−10 97 3.33 2 CX3CR1BII019 HEK293T2.9E−10 100 8.2E−10 96 2.86 2 CX3CR1BII020 HEK293T 2.2E−10 102 3.2E−1097 1.47 2

Example 7 Exploration of Linker Length and Half Life Extension

Evaluation of the Linker Length and Positioning of the Alb11 VHH

As the linker length used in a bivalent format can impact drastically onthe obtained potency, different linker lengths were evaluated.

In addition, Alb11, a Nanobody binding to human serum albumin wasincluded to increase the in vivo half-life of the formatted molecules(WO 06/122787). Different formats were made including variations on thelinker lengths used, but also the positioning of the different composingVHHs. A summary of the explored formats is shown in Table 29.

TABLE 29 Exploration of half life extension and linker length ConstructID VHH identity Linker VHH identity Linker VHH identity CX3CR1BII032CX3CR1BII66B02 9GS CX3CR1BII66B02 9GS Alb11 CX3CR1BII034 CX3CR1BII66B0235GS CX3CR1BII66B02 9GS Alb11 CX3CR1BII036 CX3CR1BII66B02 9GS Alb11 9GSCX3CR1BII66B02 CX3CR1BII040 CX3CR1BII66B02 9GS CX3CR1BII66B02 35GS Alb11CX3CR1BII041 CX3CR1BII66B02 35GS CX3CR1BII66B02 35GS Alb11 CX3CR1BII042CX3CR1BII66B02 35GS Alb11 35GS CX3CR1BII66B02

Coding sequences for the formatted VHH were cloned into an in-houseconstructed plasmid allowing expression in Pichia pastoris and secretioninto the cultivation medium. The expression vector was derived frompPICZa (Invitrogen) and contained the AOX1 promotor for tightlyregulated, methanol induced expression, a restance gene for Zeocin™, amulticloning site and the a-factor secretion signal. Upon transformationexpression cultures were grown and VHH expression was induced byaddition of methanol and allowed to continue for 48 hours at 30° C.

The potency of these different formats was evaluated using the ligandcompetition assay as described above. Seeing that the ligandconcentration used is below the EC50 value, the obtained IC50 values areequivalent to the Ki values. The obtained Ki for the different formatsis summarized in Table 30.

TABLE 30 Potency of half life extended formats in ligand competitionConstruct ID Cell line IC50 (M) % block Repeats CX3CR1BII032BA/F3-huCX3CR1 5.8E−10 99 2 CX3CR1BII034 BA/F3-huCX3CR1 5.2E−10 99 2CX3CR1BII036 BA/F3-huCX3CR1 5.6E−10 99 2 CX3CR1BII040 BA/F3-huCX3CR15.9E−10 104 1 CX3CR1BII041 BA/F3-huCX3CR1 6.4E−10 102 1 CX3CR1BII042BA/F3-huCX3CR1 8.9E−10 100 1

Impact of Human Serum Albumin on the Potency

The binding of human serum albumin (HSA) to the alb11 VHH could impacton the potency of the format and therefore ligand competition wasrepeated in presence of HSA. Briefly, to allow the binding of HSA to thealb11 VHH, the constructs under evaluation and fractalkine werepre-incubated with HSA for 30 minutes before addition to the cells. Alsothe cells were resuspended in FACS buffer supplemented with HSA. Thefinal concentration HSA used was a 50 fold excess above the highest VHHconcentration used. Subsequently, competition was allowed for 2 hoursand further processing was as described in Example 4.

Construct ID Cell line IC50 (M) % block Repeats CX3CR1BII032BA/F3-huCX3CR1 1.4E−9 100 2 CX3CR1BII034 BA/F3-huCX3CR1 1.3E−9 100 2CX3CR1BII036 BA/F3-huCX3CR1 1.4E−9 100 2

The potential interference of HSA was also evaluated in an adaptedchemotaxis setup, including HSA in the different compartments of theassay. The concentration HSA used was again a 50 fold excess over thehighest concentration of construct used and constructs were loaded withHSA for 30 minutes before start of the assay. The assay buffer was alsosupplemented with HSA such that HSA is present during the entire span ofthe experiment. As described above, the disposable ChemoTx chamber with5 μm poresize (Neuroprobe, Gaithersburg, Md., USA) was used. Cells wereharvested from an actively growing culture and washed before use inassay medium, RPMI (Gibco, Carlsbad, US) supplemented with 0.1% BSA and62.5 μM HSA (Sigma, A8763). The bottom chamber was filled with 320 pMhuman Fractalkine in a total volume of 300 μl. Upon application of themembrane, 0.13E6 cells were deposited on top of the membrane in a totalvolume of 70 μl. Chemotaxis was allowed for 3 hours at 37 C in ahumidified chamber with CO2. After this incubation period, the membranewas removed and cells in the bottom chamber were resuspended.

The amount of ATP present in well was determined using the CellTiter-Glokit (Promega, Madison Wis., USA). Read out was performed on an Envision(Perkin Elmer, Waltham, Mass., USA) with the standard factory settingsfor luminescence read out. Titration series were performed in triplicateand each plate contained control samples in triplicate as well. Ascontrol, a sample without VHH was included as well as a sample where nohuman Fractalkine was added to the bottom chamber. The obtained IC50values are listed in Table 31.

TABLE 31 Fractalkine induced chemotaxis in the presence of HSA ConstructID Cell line IC50 (M) % block Repeats CX3CR1BII032 BA/F3-huCX3CR1 6E−1098 2 CX3CR1BII034 BA/F3-huCX3CR1 9E−10 100 2 CX3CR1BII036 BA/F3-huCX3CR16E−10 102 2

Inhibition of Fractalkine Internalization by the Formatted BivalentHalf-Life Extended Polypeptides

Additional functional assays were performed to demonstrate theantagonist activity of the bivalent half-life extended polypeptides. Thepolypeptides were evaluated for their ability to inhibit theinternalization of A647-Fractalkine in CHO huCX3CR1 cells. Briefly, 1E4cells/well were plated in black clear bottom, 96 well plates (BD,Franklin Lakes, N.J., USA) and grown overnight. The cells were washedonce and then equilibrated in assay buffer (HBSS with calcium andmagnesium (Gibco) supplemented with 10 mM HEPES and 0.1% BSA). Theformatted polypeptide constructs were added and the plates wereincubated for 15 minutes at 37 C. A647-Fractalkine was then added at afinal concentration of 8 nM and the cells were incubated for 60 minutesat 37 C. The media was removed and the cells were fixed for 10 minuteswith 3.7% formaldehyde solution (Polysciences, Warrington, Pa., USA).The cells were rinsed once with PBS and the nuclei were labeled withHoechst dye (Life Technologies, Grand Island, N.Y., USA). To quantitatethe internalized labeled Fractalkine, the cells were imaged using the BDPathway bioimaging system. Image segmentation was performed byidentifying the labeled cell nucleus and drawing a 3 pixel ring aroundthat mask. Mean A647 intensity was measured in the cytoplasmic ring. Theformatted polypeptides potently inhibited Fractalkine internalization assummarized in Table 32:

TABLE 32 Inhibition of A647-Fractalkine Internalization Construct IDCell line IC50 (M) Repeats CX3CR1BII032 CHO-huCX3CR1 4.0E−10 5CX3CR1BII034 CHO-huCX3CR1 7.4E−10 3 CX3CR1BII036 CHO-huCX3CR1 4.9E−10 8CX3CR1BII040 CHO-huCX3CR1 1.0E−9 3 CX3CR1BII041 CHO-huCX3CR1 1.1E−9 2CX3CR1BII042 CHO-huCX3CR1 8.5E−10 2

An Anti-CX3CR1 Formatted Bivalent Half-Life Extended Polypeptide isDevoid of Agonist Activity

In order to confirm that a bivalent anti-CX3CR1 half-life extendedpolypeptide did not have agonist activity, CX3CR1 BII036 was evaluatedfor induction of calcium influx in the CHO huCX3CR1 cells. Fractalkinemediated increases in cytosolic calcium levels in these cells in aCX3CR1 dependent manner and CX3CR1 BII036 inhibited this response.

The CHO huCX3CR1 cells were plated at 5E4 cells/well in black clearbottom, 96 well plates (BD) and grown overnight. The cells wereincubated with Calcium-4 dye/2 mM probenicid (Molecular Devices,Sunnyvale, Calif., USA) in HBSS supplemented with 20 mM HEPES for 60minutes at 37° C. For demonstrating polypeptide antagonism, CX3CR1BII036 was preincubated with the cells for 15 minutes prior to theaddition of Fractalkine at its EC80 value. Calcium mobilization wasmonitored on a FLIPR Tetra system (Molecular Devices) as per themanufacturer's instructions. For determining agonism, there was nopreincubation with the polypeptide and instead, CX3CR1BII036 was used inplace of Fractalkine stimulation. While CX3CR1BII036 inhibitedFractalkine mediated calcium influx with an IC50 of 1.3 nM, no increasein cytosolic calcium levels were observed when the polypeptide alone wasadded at concentrations up to 1 μM.

Example 8 Exploration of Half Life Extension Formats Using Mouse Fc

To investigate alternative half-life extension modalities, the 66B02 VHHdomain was produced as a fusion protein with a mouse IgG2 Fc domain(66B02-mFc). An aspartic acid to alanine mutation (D265A) wasincorporated in the CH2 domain to abrogate potential Fc-mediatedeffector function in this construct (Baudino, J. Immunol., 181,6664-6669 (2008)). 66B02-mFc was expressed in HEK293T cells or NS0 cellsand purified by Protein A affinity chromatography followed by ionexchange chromatography. This molecule was tested for activity utilizingthe assay formats described in Example 7. The results are summarized inTable 33:

TABLE 33 66B02-mFc Activity Assay Cell line IC50 (M) Repeats LigandBA/F3-huCX3CR1 5.7E−10 2 competition Chemotaxis BA/F3-huCX3CR1 8.9E−10 3Ligand CHO-huCX3CR1 5.2E−10 5 internalization Calcium influxCHO-huCX3CR1 9.8E−10 5

While 66B02-mFc potently inhibited Fractalkine mediated CX3CR1activation, it did not display agonist activity. No increase incytosolic calcium levels was observed with treatment with up to 1 μM ofthis molecule.

Example 9 Inhibition of Plaque Progression in a Mouse AtherosclerosisModel Bivalent Half-Life Extended Polypeptides

Generation of Human CX3CR 1 Knock-In Apo E^(−/−) Mice

Given the lack of cross reactivity of the identified VHHs for mouseCX3CR1 (Example 5), a human CX3CR1 knock-in mouse line (hu CX3CR1 KI)was generated at TaconicArtemis (Koeln, Germany) to enable testing ofthese molecules in mouse disease models. A strategy was employed thatallowed the expression of the human chemokine receptor under the controlof the corresponding mouse promoter while disrupting the expression ofthe endogenous mouse protein. Briefly, a targeting vector wasconstructed where the mouse CX3CR1 coding region in exon 2 was replacedwith the complete human CX3CR1 open reading frame and flanked byselection markers and loxP sites. The targeting vector was introducedinto mouse ES cells and clones that had successfully undergonehomologous recombination were used to generate chimeric mice. These micewere bred to highly efficient Flp-deleter mice to achieve removal of theselection marker and germline transmission. The resulting hu CX3CR1 KImice in a C57BL/6 background were then crossed to Apo E^(−/−) mice (TheJackson Laboratory, Bar Harbor, Me., USA) to generate hu CX3CR1 KI ApoE^(−/−) mice. The Apo E^(−/−) mouse model provides a robust method toelicit extensive atherosclerotic plaque formation that is grosslysimilar to the human disease with respect to the site-specificlocalization of plaque formation, histological composition, and theknown risk factors (cholesterol, inflammation, hypertension, etc).

Evalulation of the Anti-CX3CR 1 Bivalent Half-Life Extended Polypeptidesin the mouse Apo E^(−/−) Atherosclerosis Model

Female hu CX3CR1 KI ApoE^(−/−) mice were fed a high fat/high cholesteroldiet containing 1.5% cholesterol for 16 weeks beginning at four weeks ofage. After 10 weeks, the animals were administered by i.p. injectionvehicle (20 nM NaCitrate pH 6.0, 115 mM NaCl), 10 mg/kg 66B02-mFc onceor twice per week or 30 mg/kg CX3CR1BII036 twice per week for 6 weeks.The animals were anesthetized by gas anaesthesia and perfused with 0.9%saline. The descending aorta to the ileac bifurcation was carefullyremoved and fixed in formalin. It was then opened longitudinally, andstained with Sudan IV for 15 minutes, followed by 70% methanol for 2minutes. The vessels were washed under running water and covered withPBS. The tissues were photographed with a digital camera using SPOTAdvanced software (SPOT Imaging Solutions, Sterling Heights, Mich.,USA). The percentage of lipid staining was determined with imageanalysis software (Image-Pro Plus, MediaCybernetics, Rockville, Md.,USA) and expressed as a percentage positive staining of the vessel. Theresults from this study are summarized in Table 34:

TABLE 34 Quantification of Plaque Size in the Descending Aorta in femalehu CX3CR1 KI Apo E^(−/−) Mice % % Reduction # Plaque in Plaque GroupDose Animals Area Area Control (10 N/A 6 3.4 N/A weeks) Control (16Vehicle 17 14.8 N/A weeks) 66B02-mFc 10 mg/kg (1xweek) 17 13.0 1666B02-mFc 10 mg/kg 17 10.3 39 (p < 0.05) (2x/week) CX3CR1BII036 30 mg/kg17 10.1 41 (p < 0.05) (2x/week)

Both 66B02-mFc and CX3CR1 BII036 significantly inhibited plaqueprogression when dosed twice weekly. This correlated with coverage asplasma levels of these molecules could be confirmed to be maintainedthroughout the study. For once weekly dosing of 66B02-mFc, detectableplasma levels were not maintained and this correlated with the lack ofsignificant efficacy observed after 6 weeks of treatment. Neithermolecule significantly affected plasma cholesterol or triglyceridelevels.

Example 10 Sequence Optimization of the Parental VHH

In general, during VHH sequence optimization, parental wild type VHHsequences are mutated to yield VHH sequences that are more identical tohuman VH3-JH germline consensus sequences. Specific amino acids in theframework regions that differ between the VHH and the human VH3-JHgermline consensus are altered to the human counterpart in such a waythat the protein structure, activity and stability are kept intact. Toinvestigate this, all sequence optimization variants were compared withthe parental VHH in three different assays: (i) determination of themelting temperature (Tm) in a Thermal Shift Assay (TSA), (ii) analysisof in vitro potency in fractalkine competition FACS, and for someconstructs (iii) analysis of in vitro potency in the fractalkine inducedchemotaxis assay.

Mutation of Framework Residues

For sequence optimization, the following mutations were investigated:E1D, S11L, A14P, E16G, R44Q, D46E, A74S, K83R and Q108L. The individualmutants that were generated in the parental sequence of CX3CR1 BII66B02are depicted in Table 35:

TABLE 35 Investigated mutations during sequence optimization of 66B02Clone number Mutations introduced C100CX3CR1BII043 A14P, A74S, K83R,Q108L C100CX3CR1BII045 E1D, A14P, A74S, K83R, Q108L C100CX3CR1BII047S11L, A14P, A74S, K83R, Q108L C100CX3CR1BII048 A14P, E16G, A74S, K83R,Q108L C100CX3CR1BII049 A14P, R44Q, A74S, K83R, Q108L C100CX3CR1BII050A14P, D46E, A74S, K83R, Q108L C100CX3CR1BII061 S11L, A14P, E16G, A74S,K83R, Q108L C100CX3CR1BII056 S11L, A14P, E16G, R44Q, A74S, K83R, Q108LC100CX3CR1BII057 S11L, A14P, E16G, D46E, A74S, K83R, Q108LC100CX3CR1BII060 S11L, A14P, E16G, R44Q, D46E, A74S, K83R, Q108L

All constructs were cloned in an E. coli expression vector, andexpressed in E. coli as myc/His-tagged proteins in a culture volume of0.25 L to 0.5 L TB medium. Expression was induced by addition of 1 mMIPTG and allowed to continue for 4 hours at 37° C. and 250 rpm. Cellswere pelleted, and periplasmic extracts were prepared by freeze-thawingand resuspension in dPBS. These extracts were used as starting materialfor immobilized metal affinity chromatography (IMAC) using Histrap FFcrude columns (GE healthcare). Nanobodies were eluted from the columnwith 250 mM imidazole and subsequently desalted towards dPBS. The purityand integrity of Nanobodies was verified by reducing SDS-PAGE.

As summarized in Table 36, A14P, A74S, K83R and Q108L mutations had noclear effect on potency as determined from competition FACS. Similarly,the additional mutations E1D, S11L and E16G did not affect potency. Theintroduction of either R44Q or D46E on the other hand resulted in asignificant drop in potency that was even more pronounced if bothmutations were introduced.

TABLE 36 Potency of sequence optimization constructs determined byligand competition FACS Clone number IC50 % block Tm at pH 7CX3CR1BII66B02 2.6E−09 101.0 65.66 C100CX3CR1BII043 2.2E−09 101 66.49C100CX3CR1BII045 2.2E−09 101.2 66.07 C100CX3CR1BII047 2.3E−09 101.266.49 C100CX3CR1BII048 1.9E−09 101.2 67.74 C100CX3CR1BII049 1.8E−08101.2 66.07 C100CX3CR1BII050 1.7E−08 101.1 71.90 C100CX3CR1BII0611.4E−09 98.9 68.57 C100CX3CR1BII056 1.6E−08 101.1 68.57 C100CX3CR1BII0571.4E−08 99.4 74.39 C100CX3CR1BII060 1.9E−07 98.4 74.81

Also the melting temperature, predictive for the stability of the VHH,was evaluated. Most individual mutations had limited to no effect exceptfor the D46E mutation which raised the melting temperature byapproximately 6° C. The introduction of the combined mutations alsoenhanced the thermal stability, cfr 057 and 060.

Due to the major effects on the potency in ligand competition FACS, themutations R44Q and D46E were not included in the final sequence.

Mutation of CDR Residues

Based on the in silico analysis of the parental sequence, aglycosylation site was predicted at position 52. Therefore two librarieswere constructed; one for position 52 and one for position 53, which wasdesigned to include all possible amino acids at the respective position.The libraries were screened as periplasmic extracts in a ligandcompetition FACS. First, a dilution series was made of periplasmicmaterial from the parental sequence and three dilutions were selectedfor further screening. A first dilution point (two fold) was chosen togive full block of the ligand interaction whereas the other two dilutionpoints (128 and 512 fold) should result in 70% and 40% blockrespectively. Upon production of the periplasmic extracts from thelibrary, all samples were split in two and one of them was subjected toa heat treatment. Both the non-treated and the heat treated samples weresubsequently analyzed in the ligand competition FACS at the threedilution points. The impact of the mutation could be estimated bycomparing the obtained blockage with that from the parental sequence.The analysis of the heat treated samples provides a measure for apotential impact on stability of the mutation.

Based upon the initial screening results, seven mutations were selectedfor further characterization. The obtained potency in ligand competitionFACS is shown in Table 37.

TABLE 37 Removal glycosylation site at position 52 Construct IC50 (M) %block Tm at pH 7 C100CX3CR1BII66B02 2.5E−09 98.0 65.66 CX3CR1BII66B02(N52S, Q108L) 1.7E−09 98.0 66.07 CX3CR1BII66B02 (N52Q, Q108L) 2.1E−0997.9 59.83 CX3CR1BII66B02 (N52G, Q108L) 1.1E−09 98.0 59.83CX3CR1BII66B02 (N52T, Q108L) 2.8E−09 98.0 66.07 CX3CR1BII66B02 (S53T,Q108L) 1.3E−09 98.1 66.07 CX3CR1BII66B02 (S53G, Q108L) 1.2E−09 98.364.83 CX3CR1BII66B02 (S53P, Q108L) 8.0E−10 98.2 66.91

From this analysis, sequence alignment with the human reference sequenceand based upon an in silico T cell epitope recognition predictionprogram, it was decided to include the mutations N52S and S53T in thesequence.

Because of stability reasons an additional library was made for position32.The ligand competition screening was set up in a similar fashion asdescribed above. Again three dilutions of the periplasmic extracts werescreened and the obtained % block was compared with that obtained forthe parental sequence. Upon analysis of the various mutants, thesubstitution of N32T was chosen and included in the final sequenceoptimized variant.

Example 11 Analysis of the Optimized Variants

In a final characterization round the constructs listed in Table 38 werecharacterized.

TABLE 38 Sequence optimized variants of the lead VHH 66B02 Clone numberMutation introduced SEQ ID NO: CX3CR1BII00306 CX3CR1BII66B02(E1D, S11L,A14P, E16G, N32T, N52S, 138 A74S, K86R, Q113L) CX3CR1BII00307CX3CR1BII66B02(E1D, S11L, A14P, E16G, N32T, N52S, 139 S53T, A74S, K86R,Q113L) CX3CR1BII00308 CX3CR1BII66B02(E1D, S11L, A14P, E16G, A74S, K86R,140 Q113L) CX3CR1BII00312 CX3CR1BII66B02(E1D, S11L, A14P, E16G, N32T,N52S, 225 A74S, K86R, Q113L)-9GS-Alb11-9GS- CX3CR1BII66B02(S11L, A14P,E16G, N32T, N52S, A74S, K86R, Q113L) CX3CR1BII00313 CX3CR1BII66B02(E1D,S11L, A14P, E16G, N32T, N52S, 226 S53T, A74S, K86R,Q113L)-9GS-Alb11-9GS- CX3CR1BII66B02(S11L, A14P, E16G, N32T, N52S, S53T,A74S, K86R, Q113L) CX3CR1BII00314 CX3CR1BII66B02(E1D, S11L, A14P, E16G,A74S, K86R, 227 Q113L)-9GS-Alb11-9GS- CX3CR1BII66B02(S11L, A14P, E16G,A74S, K86R, Q113L)

A competition FACS experiment was performed as described above as wellas a determination of the melting temperature. The obtained values arerepresented in Table 39.

TABLE 39 Competition FACS and Tm of the sequence optimized variantsConstruct IC50 (M) % block Tm at pH 7 C100CX3CR1BII66B02 2.5E−09 98.065.05 CX3CR1BII00306 1.7E−09 97.0 68.54 CX3CR1BII00307 1.9E−09 97.068.13 CX3CR1BII00308 1.6E−09 97.0 68.13 CX3CR1BII00312 4.6E−10 100.059.37 CX3CR1BII00313 4.0E−10 100.0 58.88 CX3CR1BII00314 6.5E−10 100.058.40

These constructs were also characterized in fractalkine inducedchemotaxis as described above (Table 40).

TABLE 40 Ligand induced chemotaxis with sequence optimized variantsConstruct IC50 (M) % block n C100CX3CR1BII66B02 3.6E−08 91 3CX3CR1BII00306 6.3E−08 95 2 CX3CR1BII00307 6.3E−08 100 2 CX3CR1BII003084.4E−08 89 2 CX3CR1BII00312 2.7E−09 99 3 CX3CR1BII00313 2.7E−09 99 3CX3CR1BII00314 3.6E−09 100 3

Selected constructs were evaluated for inhibition of A647-Fractalkineinduced internalization in CHO huCX3CR1 cells. The results aresummarized in Table 41:

TABLE 41 Ligand induced internalization with sequence optimized variantsConstruct IC50 (M) n CX3CR1BII00312 5.5E−10 1 CX3CR1BII00313 3.3E−10 6

A Sequence Optimized Anti-CX3CR1 Half-Life Eextended Polypeptide isDevoid of Agonist Activity

In order to confirm that sequence optimized anti-CX3CR1 half-lifeextended polypeptide does not have agonist activity, CX3CR1BII00313 wasevaluated for induction of calcium influx in the CHO huCX3CR1 cells.While preincubation with CX3CR1BII00313 inhibited Fractalkine-mediatedcalcium influx with an IC50 of 1.3 nM, no increase in cytosolic calciumlevels were observed when the polypeptide alone was added atconcentrations up to 1 μM.

Example 12 Exploration of Half Life Extension Formats Using Human Fc

To investigate additional half-life extension modalities, theCX3CR1BII00306 and CX3CR1BII00307 sequence optimized VHH domains wereproduced as fusion proteins with a human IgG1 Fc domain (306D-hFc and307D-hFc). Two mutations were incorporated in the CH2 domain to abrogatepotential Fc-mediated effector function in this construct. 306D-hFc and307D-hFc were expressed in HEK293T cells or NS0 cells and purified byProtein A affinity chromatography followed by ion exchangechromatography. These molecules were tested for functional activityutilizing the assay formats described in Example 7. The results aresummarized in Table 42:

TABLE 42 Activity of hFc Fusion Proteins 306D- 307D- 307D- 306D-hFc hFchFc hFc Assay Cell line IC50 Repeats IC50 Repeats Ligand BA/F3- 6.9E−102 7.0E−10 2 competition huCX3CR1 Chemotaxis BA/F3- 2.9E−9 2 3.0E−9 3huCX3CR1 Ligand CHO- 4.8E−10 3 3.7E−10 3 internalization huCX3CR1Calcium influx CHO- 1.3E−9 3 3.2E−9 3 huCX3CR1

While these molecules potently inhibited Fractalkine mediated CX3CR1activation, they did not display agonist activity. No increases incytosolic calcium levels were observed with treatment with up to 1 μM ofthese Nanobodies alone.

Example 13 Inhibition of Plaque Progression in a Mouse AtherosclerosisModel by a Sequence Optimized Anti-CX3CR1 Nanobody

Female hu CX3CR1 KI ApoE^(−/−) mice were fed a high fat/high cholesteroldiet containing 1.5% cholesterol for 16 weeks beginning at four weeks ofage. After 10 weeks, the animals were administered by i.p. injectionvehicle (20 mM NaCitrate pH 6,0, 115 mM NaCl), 30 mg/kg CX3CR1BII00313once or twice per week or 30 mg/kg CX3CR1BII036 twice per week for 6weeks. The animals were sacrificed and the percentage of plaque area inthe descending aorta was quantitated as described above. The resultsfrom this study are summarized in Table 43:

TABLE 43 Quantification of Plaque Size in the Descending Aorta in femalehu CX3CR1 KI Apo E^(−/−) Mice % Reduction % Plaque in Plaque Group Dose# Animals Area Area Control (10 N/A 6 2.1 N/A weeks) Control (16 Vehicle18 12.0 N/A weeks) CX3CR1BII00313 30 mg/kg 17 10.7 13 (1xweek)CX3CR1BII00313 30 mg/kg 18 5.9 62 (p < 0.01) (2x/week) CX3CR1BII036 30mg/kg 17 6.8 52 (p < 0.01) (2x/week)

Both CX3CR1BII00313 and CX3CR1BII036 significantly inhibited plaqueprogression when dosed twice weekly. This correlated with coverage asplasma levels of these molecules could be confirmed to be maintainedthroughout the study. For once weekly dosing of CX3CR1BII00313,detectable plasma levels were not maintained and this correlated withthe lack of significant efficacy observed after 6 weeks of treatment.Neither molecule significantly affected plasma cholesterol ortriglyceride levels.

Example 14 Nanobody Binding to Primary Human and Cynomolgus MonkeyCD14+Cells in Whole Blood

Competition FACS with formatted sequence optimized anti-CX3CR1 Nanobody

To confirm binding of the formatted sequence optimized anti-CX3CR1Nanobody to human primary cells, CX3CR1BII00313 was demonstrated tocompete for the binding of A647 labeled CX3CR1BII018 (A647-018) to CD14+cells in a competition FACS assay in whole blood. Briefly, a mouseanti-human CD14 antibody conjugated with eFluor 450 (eBioscience, SanDiego, Calif., USA) was diluted 1:10 in EDTA treated whole blood from ahealthy human donor. 40 μl/well was added to 96 well polystyrene roundbottom plate followed by 10 μl/well of CX3CR1BII00313 diluted in StainBuffer with BSA (BD Pharmingen) at a final concentration ranging from100 nM to 0.002 pM and the samples were incubated for 20 minutes at roomtemperature. 10 μl/well of A647-018 in Stain Buffer was then added toyield a final concentration of 1 nM (the EC80 of A647-018 binding) andthe samples were incubated for an additional 20 minutes at roomtemperature. 220 μl/well of 1-Step Fix/Lyse solution (eBioscience) wasthen added. After a 10 minute room temperature incubation the cells werepelleted, washed twice in Stain buffer and resuspended in this buffer.The samples were analyzed on a BD LSR II flow cytometer. The medianfluorescence intensity for AlexaFluor 647 was quantified for the gateCD14 positive cell population. CX3CR1BII00313 potently inhibited thebinding of A647-018 to CD14 positive cells in human blood with an IC50of 0.35 nM (n=8).

To confirm binding of the formatted sequence optimized anti-CX3CR1Nanobody to cynomolgus monkey primary cells, CX3CR1BII00313 wasdemonstrated to compete for the binding of A647 labeled CX3CR1BII018(A647-018) to CD14+ cells in a competition FACS assay in cynomolgusmonkey whole blood. The method used was analogous to that outlined aboveexcept the final concentration of A647-018 was 3 nM (the EC80 ofA647-018 binding) and ACK lysing buffer (Life Technologies) was usedinstead of the 1-Step Fix/Lyse solution. The cells were resuspended inStain buffer supplemented with 1% formaldehyde prior to analysis.CX3CR1BII00313 potently inhibited the binding of A647-018 to CD14positive cells in cynomolgus monkey blood with an IC50 of 0.43 nM (n=4).

Example 15 Pharmacokinetics (PK) in Cynomolgus Monkeys

A pharmacokinetic study was conducted in naïve male cynomolgus monkeys(Macaca fascicularis) 2 -5 years of age with a body weight range between2.4-3.5 kg. The monkeys were divided into four treatment groups. Group 1(n=3) received 0.2 mg/kg of CX3CR1BII00313 i.v.; Group 2 (n=3) received2 mg/kg of CX3CR1BII00313 i.v.; Group 3 (n=3) received 2 mg/kgCX3CR1BII00313 s.c. and Group 4 (n=3) received 5 mg/kg CX3CR1BII00313i.v. CX3CR1BII00313 was administered as a 2 mg/ml solution in citratebuffer (20 mM sodium citrate/115 mM sodium chloride, pH 6.0). Bloodsamples were collected over 6 weeks from a peripheral vein into serumseparator tubes for PK analysis.

Serum samples were analyzed using a MSD (Meso Scale Discovery) format.Briefly, a biotinylated anti-Nanobody antibody was bound to a MSDstandard streptavidin plate (Meso Scale Discovery, Rockville, Md., USA).The plates were washed with 0.05% Tween 20 in phosphate buffered salineand blocked with 5% w/v of SeraCare BSA (SeraCare Life Sciences,Milford, Mass., USA) prior to incubation with serum samples.CX3CR1BII00313 was detected utilizing a sulfo-labeled anti-NanobodyNanobody and the plates were analyzed on a Sector Imager 2400 (MesoScale Discovery). Varying concentrations of CX3CR1BII0313 from 5000 to0.5 ng/ml in 5% monkey serum were used as standards. Target engagementwas assessed by monitoring levels of free CX3CR1 on CD14+gatedmonocytes. This assay was analogous to the competition FACS assaysummarized in Example 14 except no additional CX3CR1BII00313 was added.Serum samples were also monitored for the presence of primate anti-humanantibodies (PAHA) as they may impact assessment of PK and free CX3CR1.

ForteBio RED96 was used for detection of PAHA. Briefly, biotinylatedCX3CR1BII0313 was captured over streptavidin sensors. Pooled naïvemonkey serum was then used as a negative control to calculate cut-offvalue (defined as two fold above the average binding signal of naïvesera). All serum samples were diluted 20 fold in buffer and the PAHAresponse was determined to be positive if the binding signal was greaterthan the cut-off value.

Data for time points following detection of PAHA were excluded fromPK/PD analysis. The PK data are summarized in Table 44 below.

TABLE 44 Pharmacokinetic parameters of CX3CR1BII00313 Dose normalized CLT_(1/2) MRT AUC(0-14 d) Dose (mg/kg) (mL/day/kg) (day) (day) (nM · d) F% IV 0.2 113 1 1 ** IV 2.0 9 ± 1 9 ± 2 8 ± 2 56530 IV 5.0 ** ** ** 58604SC 2.0 54 ** insufficient data for characterization of terminal phase

Clearance and half-life at 2.0 mg/kg i.v. were 9.4 mL/d/kg and 9.6 days,respectively. At 0.2 mg/kg i.v., clearance was substantially higher (113mL/d/kg) consistent with saturable target-mediated disposition (TMD)pharmacokinetics. Dose-adjusted AUC_((0-14d)) was comparable between the2 and 5 mg/kg i.v. doses suggesting saturation of TMD at the 2 mg/kgdose. Exposure at 2 weeks following either i.v. or s.c. Nanobodyadministration was >70 nM and bioavailability after s.c. administrationwas 54%. Free receptor tracked with exposure with greater than 90%target coverage maintained at exposures >10 nM.

The invention claimed is:
 1. A polypeptide comprising an anti-CX3CR1 (anti-CX3 chemokine receptor 1) immunoglobulin single variable domain, wherein said polypeptide comprises a CDR1, CDR2 and CDR3 having the amino acid sequences set forth in: SEQ ID No: 141, 162 and 186, respectively; or SEQ ID No: 141, 163 and 187, respectively; or SEQ ID No: 141, 164 and 186, respectively; or SEQ ID No: 141, 166 and 186, respectively; or SEQ ID No: 141, 167 and 186, respectively; or SEQ ID No: 141, 167 and 189, respectively; or SEQ ID No: 141, 168 and 186, respectively; or SEQ ID No: 141, 168 and 187, respectively; or SEQ ID No: 141, 169 and 190, respectively; or SEQ ID No: 141, 170 and 186, respectively; or SEQ ID No: 141, 171 and 186, respectively; or SEQ ID No: 141, 174 and 186, respectively; or SEQ ID No: 141, 175 and 187, respectively; or SEQ ID No: 142, 165 and 188, respectively; or SEQ ID No: 142, 173 and 188, respectively; or SEQ ID No: 143, 164 and 186, respectively; or SEQ ID No: 144, 172 and 187, respectively; or SEQ ID No: 145, 172 and 187, respectively; or SEQ ID No: 141, 214 and 186, respectively; or SEQ ID No: 141, 215 and 186, respectively; or SEQ ID No: 141, 216 and 186, respectively; or SEQ ID No: 141, 217 and 186, respectively; or SEQ ID No: 141, 218 and 186, respectively; or SEQ ID No: 141, 219 and 186, respectively; or SEQ ID No: 141, 220 and 186, respectively; or SEQ ID No: 213, 221 and 186, respectively; or SEQ ID No: 213, 214 and 186, respectively.
 2. The polypeptide according to claim 1, wherein the polypeptide comprises a CDR1, CDR2 and CDR3 having amino acid sequences set forth in: SEQ ID NO's: 141, 164 and 186, respectively, SEQ ID NO's: 141, 162 and 186, respectively, SEQ ID NO's: 213, 214 and 186 respectively, or SEQ ID NO's: 213, 221 and 186 respectively.
 3. The polypeptide according to claim 1, wherein said anti-CX3CR1 immunoglobulin single variable domain is a VHH domain comprising the sequence set forth in: a) the amino acid sequence of SEQ ID NO: 3; b) an amino acid sequence that has at least 90% amino acid identity with the amino acid sequences of SEQ ID NO: 3; or c) an amino acid sequence of any one of SEQ ID NO: 1-48, 121-140 or 222-224.
 4. The polypeptide according to claim 1, wherein said anti-CX3CR1 immunoglobulin single variable domain comprises the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 121-140 or SEQ ID NO: 222-224.
 5. The polypeptide according to claim 1, wherein the polypeptide further comprises a half-life extending moiety.
 6. The polypeptide according to claim 5, wherein said half-life extending moiety is covalently linked to said polypeptide and is selected from the group consisting of an albumin binding moiety, such as an anti-albumin immunoglobulin domain, a transferrin binding moiety, such as an anti-transferrin immunoglobulin domain, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of human serum albumin, an albumin binding peptide or a Fc domain.
 7. The polypeptide according to claim 5, wherein said half-life extending moiety consists of an anti-albumin immunoglobulin single variable domain.
 8. The polypeptide according to claim 7, wherein the immunoglobulin single variable domain is selected from a VHH domain, a humanized VHH domain, a camelized VH domain, a domain antibody, a single domain antibody and/or “dAb”s.
 9. The polypeptide according to claim 8, wherein the anti-albumin immunoglobulin single variable domain comprises a sequence selected from any one of SEQ ID NO's: 230-232.
 10. The polypeptide according to claim 1, wherein said polypeptide further comprises a second immunoglobulin single variable domain.
 11. The polypeptide according to claim 10 wherein said second immunoglobulin single variable domain comprises a second anti-CX3CR1 immunoglobulin single variable domain.
 12. The polypeptide according to claim 11, wherein said second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 amino acid sequences set forth in SEQ ID No: 141, 162 and 186, respectively; or SEQ ID No: 141, 163 and 187, respectively; or SEQ ID No: 141, 164 and 186, respectively; or SEQ ID No: 141, 166 and 186, respectively; or SEQ ID No: 141, 167 and 186, respectively; or SEQ ID No: 141, 167 and 189, respectively; or SEQ ID No: 141, 168 and 186, respectively; or SEQ ID No: 141, 168 and 187, respectively; or SEQ ID No: 141, 169 and 190, respectively; or SEQ ID No: 141, 170 and 186, respectively; or SEQ ID No: 141, 171 and 186, respectively; or SEQ ID No: 141, 174 and 186, respectively; or SEQ ID No: 141, 175 and 187, respectively; or SEQ ID No: 142, 165 and 188, respectively; or SEQ ID No: 142, 173 and 188, respectively; or SEQ ID No: 143, 164 and 186, respectively; or SEQ ID No: 144, 172 and 187, respectively; or SEQ ID No: 145, 172 and 187, respectively; or SEQ ID No: 141, 214 and 186, respectively; or SEQ ID No: 141, 215 and 186, respectively; or SEQ ID No: 141, 216 and 186, respectively; or SEQ ID No: 141, 217 and 186, respectively; or SEQ ID No: 141, 218 and 186, respectively; or SEQ ID No: 141, 219 and 186, respectively; or SEQ ID No: 141, 220 and 186, respectively; or SEQ ID No: 213, 221 and 186, respectively; or SEQ ID No: 213, 214 and 186, respectively.
 13. The polypeptide according to claim 12, wherein said first and said second immunoglobulin single variable domains comprise the same CDR1, CDR2 and CDR3.
 14. The polypeptide according to claim 13, wherein said first and second immunoglobulin single variable domains comprise a VHH domain having an amino acid sequence set forth in a) the amino acid sequence of SEQ ID NO: 3; b) an amino acid sequence that has at least 90% amino acid identity with the amino acid sequences of SEQ ID NO: 3; c) an amino acid sequence of any one of SEQ ID NO: 1-48, 121-140 or 222-224; d) the amino acid sequence of SEQ ID NO: 49; e) an amino acid sequence that has at least 95% amino acid identity with the amino acid sequences of SEQ ID NO: 49; f) an amino acid sequence of any one of SEQ ID NO: 49-52; g) the amino acid sequence of SEQ ID NO: 67; h) an amino acid sequence that has at least 90% amino acid identity with the amino acid sequences of SEQ ID NO: 67; or i) an amino acid sequence of any one of SEQ ID NO: 53-120.
 15. The polypeptide according to claim 14, wherein said first and said second immunoglobulin single variable domains comprise the same VHH domain.
 16. The polypeptide according to claim 10, wherein said first and second immunoglobulin single variable domains are a VH, VL, VHH, camelized VH or VHH.
 17. A polypeptide comprising a first immunoglobulin single variable domain and a second immunoglobulin single variable domain, each comprising CDR1, CDR2 and CDR3 having amino acid sequences set forth in SEQ ID NO's: 141, 164 and 186, or SEQ ID NO's: 141, 162 and 186, or SEQ ID NO's: 213, 214 and 186, or SEQ ID NO's: 213, 221 and
 186. 18. A polypeptide comprising a first immunoglobulin single variable domain and a second immunoglobulin single variable domain, wherein each immunoglobulin single variable domain is a VHH domain comprising the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, or any of SEQ ID NO: 121-140 or 222-224.
 19. The polypeptide according to claim 18, wherein the polypeptide further comprises a half-life extending moiety.
 20. The polypeptide according to claim 19, wherein said half-life extending moiety is covalently linked to said polypeptide and is selected from the group consisting of an albumin binding moiety, such as an anti-albumin immunoglobulin domain, a transferrin binding moiety, such as an anti-transferrin immunoglobulin domain, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of human serum albumin, an albumin binding peptide or a Fc domain.
 21. The polypeptide according to claim 19, wherein said half-life extending moiety consists of an anti-albumin immunoglobulin single variable domain.
 22. The polypeptide according to claim 21, wherein the immunoglobulin single variable domain is selected from a VHH domain, a humanized VHH domain, a camelized VH domain, a domain antibody, a single domain antibody and/or “dAb”s.
 23. The polypeptide according to claim 22, wherein the anti-albumin immunoglobulin single variable domain is selected from SEQ ID NO's: 230-232.
 24. A polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 225-227 or 257-262.
 25. A pharmaceutical composition comprising (i) a polypeptide according to claim 1, and (ii) a pharmaceutically acceptable carrier, and optionally (iii) a diluent, excipient, adjuvant and/or stabilizer.
 26. The pharmaceutical composition according to claim 25, wherein said pharmaceutical composition is suitable for intravenous or subcutaneous injection in a human being.
 27. A polypeptide comprising the amino acid sequence of SEQ ID NO:
 225. 28. A polypeptide comprising the amino acid sequence of SEQ ID NO:
 226. 29. A polypeptide comprising the amino acid sequence of SEQ ID NO:
 227. 30. A polypeptide comprising the amino acid sequence of SEQ ID NO:
 258. 31. A polypeptide comprising the amino acid sequence of SEQ ID NO:
 261. 32. A pharmaceutical composition comprising (i) a polypeptide according to claim 27, and (ii) a pharmaceutically acceptable carrier, and optionally (iii) a diluent, excipient, adjuvant and/or stabilizer.
 33. A pharmaceutical composition comprising (i) a polypeptide according to claim 28, and (ii) a pharmaceutically acceptable carrier, and optionally (iii) a diluent, excipient, adjuvant and/or stabilizer.
 34. A pharmaceutical composition comprising (i) a polypeptide according to claim 29, and (ii) a pharmaceutically acceptable carrier, and optionally (iii) a diluent, excipient, adjuvant and/or stabilizer.
 35. A pharmaceutical composition comprising (i) a polypeptide according to claim 30, and (ii) a pharmaceutically acceptable carrier, and optionally (iii) a diluent, excipient, adjuvant and/or stabilizer.
 36. A pharmaceutical composition comprising (i) a polypeptide according to claim 31, and (ii) a pharmaceutically acceptable carrier, and optionally (iii) a diluent, excipient, adjuvant and/or stabilizer. 